Flowcells with linear waveguides

ABSTRACT

For example, a flowcell includes: a nanowell layer having a first set of nanowells and a second set of nanowells to receive a sample; a first linear waveguide associated with the first set of nanowells, and a second linear waveguide associated with the second set of nanowells; and a first grating for the first linear waveguide, and a second grating for the second linear waveguide, the first and second gratings providing differential coupling of first light and second light.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application a continuation application of U.S. patent applicationSer. No. 17/255,765, filed Dec. 23, 2022, which is a 35 U.S.C. § 371National Stage of International Patent Application No.PCT/US2020/070063, filed May 19, 2020, which itself claims the benefitof and priority to U.S. Provisional Patent Application No. 62/868,423,filed on Jun. 28, 2019, the content of each of which is incorporated byreference herein in its entirety and for all purposes.

BACKGROUND

Samples of different materials can be analyzed using one or more of avariety of analytical processes. For example, sequencing such ashigh-throughput DNA sequencing can be the basis for genomic analysis andother genetic research. For example, sequencing by synthesis (SBS)technology uses modified deoxyribonucleotide triphosphates (dNTPs)including a terminator and a fluorescent dye having an emissionspectrum. In this and other types of sequencing, characteristics of asample of genetic material are determined by illuminating the sample,and by detecting emission light (e.g., fluorescent light) that isgenerated in response to the illumination.

It may be desirable to ensure good quality of the analysis of the sampleas well as to facilitate that the analysis is performed at relativelyhigh speed. For example, the amount of the sample material that isanalyzed at each individual stage drives the resulting throughput of theanalysis process. It may be attempted to distribute the sample materialmore densely in the analysis equipment to allow more material to beanalyzed at any given time. However, characteristics of the analysissystem such as the maximum resolution available from imaging optics maylimit the extent to which such an approach can increase the throughput.

SUMMARY

In a first aspect, a flowcell includes: a nanowell layer having a firstset of nanowells and a second set of nanowells to receive a sample; afirst linear waveguide associated with the first set of nanowells, and asecond linear waveguide associated with the second set of nanowells; anda first grating for the first linear waveguide, and a second grating forthe second linear waveguide, the first and second gratings providingdifferential coupling of first light and second light.

Implementations can include any or all of the following features. Thefirst and second gratings are spatially offset from each other. Thefirst and second linear waveguides are positioned adjacent to eachother, the flowcell further comprising: a third linear waveguidepositioned adjacent to the second linear waveguide opposite from thefirst linear waveguide. The third linear waveguide shares the firstgrating with the first linear waveguide. The flowcell further comprisesa third grating for the third linear waveguide. The third grating hasthe same spatial offset from the second grating as has the firstgrating. The third grating is spatially offset from each of the firstand second gratings. The first grating is positioned toward a first endof the first linear waveguide, wherein the second grating is positionedtoward a second end of the second linear waveguide, and wherein thefirst end is positioned opposite from the second end. The first gratingis positioned on a triangular substrate. The first and second gratingshave different grating periods from each other. The first and secondlinear waveguides are positioned adjacent each other, the flowcellfurther comprising: a third linear waveguide positioned adjacent to thesecond linear waveguide opposite from the first linear waveguide; and athird grating for the third linear waveguide. The third grating has thesame grating period as the first grating. The third grating has agrating period different from each of the grating periods of the firstand second gratings. The nanowells in at least one of the first andsecond sets of nanowells have a spacing from each other that isresolvable according to a resolution distance of emission optics for theflowcell. The first and second linear waveguides are positioned closerto each other than the resolution distance of the emission optics. Thedifferential coupling of the first light comprises coupling the firstlight into the first linear waveguide and minimizing coupling of thefirst light into the second linear waveguide. The differential couplingof the second light comprises coupling the second light into the secondlinear waveguide and minimizing coupling of the second light into thefirst linear waveguide. The differential coupling is at least in partdue to a coupler parameter of one or more of the first grating or thesecond grating. The coupler parameter comprises at least one selectedfrom the group consisting of: a refractive index, a pitch, a groovewidth, a groove height, a groove spacing, a grating non-uniformity, agroove orientation, a groove curvature, a coupler shape, andcombinations thereof. The differential coupling is at least in part dueto a waveguide parameter of one or more of the first linear waveguide orthe second linear waveguide. The waveguide parameter comprises at leastone selected from the group consisting of: a cross sectional profile, arefractive index difference, a mode matching, and combinations thereof.The first and second sets of nanowells are arranged in a polygonalarray. The polygonal array comprises a rectangular array or a hexagonalarray. The first and second sets of nanowells are arranged in thehexagonal array, which forms at least one hexagon, the hexagonincluding: first and second nanowells of the first set of nanowells, thefirst and second nanowells being part of a first row of nanowells thatextends along the first linear waveguide; third, fourth and fifthnanowells of the second set of nanowells, the third, fourth and fifthnanowells being part of a second row of nanowells that extends along thesecond linear waveguide; and sixth and seventh nanowells of a third setof nanowells, the sixth and seventh nanowells being part of a third rowof nanowells that extends along a third linear waveguide. The first setof nanowells comprises a first row of nanowells, and wherein the secondset of nanowells comprises a second row of nanowells. Each of the firstand second rows of nanowells is aligned with at least one of the firstand second linear waveguides. The first row of nanowells extends alongthe first linear waveguide, wherein the second row of nanowells extendsalong the second linear waveguide, wherein the first linear waveguide isparallel and adjacent to the second linear waveguide, and wherein thefirst row of nanowells is in phase with the second row of nanowells, theflowcell further comprising: a third linear waveguide that is paralleland adjacent to the second linear waveguide; and a third row ofnanowells extending along the third linear waveguide, wherein the thirdrow of nanowells is out of phase with the first and second rows ofnanowells. The flowcell further comprises: a fourth linear waveguidethat is parallel and adjacent to the third linear waveguide; and afourth row of nanowells extending along the fourth linear waveguide,wherein the fourth row of nanowells is in phase with the third row ofnanowells. The first and second linear waveguides are parallel andadjacent each other, wherein the first set of nanowells comprises firstand second rows of nanowells extending along the first linear waveguideon opposite sides thereof, and wherein the second set of nanowellscomprises third and fourth rows of nanowells extending along the secondlinear waveguide on opposite sides thereof. At least one nanowell of thefirst and second sets of nanowells has a non-circular opening. Thenon-circular opening comprises an elliptical opening. The flowcellfurther comprises structure between the first and second linearwaveguides to reduce cross-coupling. The structure comprises a series ofblocks. The structure provides refractive indices that alternate alongthe structure. The first linear waveguide and the first grating arepositioned in a first layer of the flowcell, wherein the second linearwaveguide and the second grating are positioned in a second layer of theflowcell, wherein the first and second sets of nanowells are positionedin a third layer of the flowcell, and wherein the second layer ispositioned further from the third layer than is the first layer.

In a second aspect, a method comprises: applying, at a flowcell, asample to a first set of nanowells and to a second set of nanowells;differentially coupling, using a first grating, first light into atleast a first linear waveguide associated with the first set ofnanowells; and differentially coupling, using a second grating, secondlight into at least a second linear waveguide associated with the secondset of nanowells.

Implementations can include any or all of the following features. Thefirst and second gratings are spatially offset from each other, themethod further comprising controlling an illumination componentregarding at least one of the first light or the second light.Controlling the illumination component comprises controlling a beamparameter of a light beam generating at least one of the first light orthe second light. Controlling the beam parameter comprises at least oneselected from the group consisting of: controlling a location of thelight beam, controlling an angle of incidence of the light beam,controlling a divergence of the light beam, controlling a mode profileof the light beam, controlling a polarization of the light beam,controlling an aspect ratio of the light beam, controlling a diameter ofthe light beam, controlling a wavelength of the light beam, andcombinations thereof. The first light is being differentially coupledduring a first scan performed across the flowcell in a first scandirection, and the second light is being differentially coupled during asecond scan performed across the flowcell in a second scan directionopposite to the first scan direction. The first and second gratings havedifferent grating periods from each other, the method further comprisingarranging an illumination component so that the first light isdifferentially coupled, and arranging the illumination component so thatthe second light is differentially coupled. The first and second linearwaveguides are positioned adjacent each other, and wherein the flowcellfurther comprises a third linear waveguide positioned adjacent to thesecond linear waveguide opposite from the first linear waveguide. Theflowcell further comprises a third grating for the third linearwaveguide. The method further comprises differentially coupling thefirst light also into the third linear waveguide using the thirdgrating. The method further comprises differentially coupling thirdlight into at least the third linear waveguide using the third grating.The third linear waveguide shares the first grating with the firstlinear waveguide. The nanowells in at least one of the first and secondsets of nanowells have a spacing from each other that is resolvableaccording to a resolution distance of emission optics for the flowcell.The first and second linear waveguides are positioned closer to eachother than the resolution distance of the emission optics.Differentially coupling the first light comprises coupling the firstlight into the first linear waveguide and minimizing coupling of thefirst light into the second linear waveguide. Differentially couplingthe second light comprises coupling the second light into the secondlinear waveguide and minimizing coupling of the second light into thefirst linear waveguide.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a cross section of part of an example flowcell with linearwaveguides.

FIGS. 2A-2B illustrate examples with a flowcell having staggeredgratings.

FIGS. 3A-3B illustrate examples with a flowcell having gratings withdifferent grating periods.

FIG. 4 shows another example of a flowcell having staggered gratings.

FIG. 5 shows a cross section of part of an example flowcell.

FIG. 6 shows an example of a flowcell where multiple linear waveguidesshare a common grating.

FIG. 7 is a diagram of an example illumination system.

FIGS. 8-9 are flowcharts of example methods.

FIG. 10A shows an example of a hexagonal array of non-circularnanowells.

FIG. 10B shows an example of a triangular array of circular nanowells.

FIG. 11 shows another example of a flowcell having staggered gratings.

FIG. 12 shows another example of a flowcell having staggered gratings.

FIG. 13 shows another example of a flowcell having staggered gratings.

FIG. 14 schematically shows a light beam impinging on a surface.

FIGS. 15A-15C show examples of gratings.

FIG. 16 shows examples of shapes of couplers.

FIG. 17 shows examples of cross-sectional profiles for linearwaveguides.

FIG. 18 shows a cross section of part of another example flowcell withlinear waveguides.

FIG. 19 is a flowchart of an example method.

DETAILED DESCRIPTION

The present disclosure describes systems, techniques, articles ofmanufacture, and/or compositions of matter that facilitate improvedanalysis of samples. In some implementations, differential coupling canbe provided into two or more linear waveguides. For example, being ableto differentially couple light into linear waveguides can allow asubstrate (e.g., a layer of nanowells for holding sample material) tohave an increased density of the sample material. In someimplementations, one or more parameters regarding the analysis systemand/or the process can be selected or adjusted so as to obtaindifferential coupling. For example, such parameter(s) can include one ormore beam parameters, one or more coupler parameters, one or morewaveguide parameters, or combinations thereof.

In some implementations, analysis imaging can be performed upon samplematerial having an increased density of distribution on a substrate,which can increase the throughput of the analysis process. For example,sample material can be distributed with a density where individualportions of the sample are positioned at closer distances from eachother than can be resolved using the available imaging technology, suchas microscopy equipment. An analysis process may selectively image onlyfirst portions of the sample at a time, and not image second portionsnear the first portions, and subsequently image the second portionswithout (again) imaging the first portions. Such an approach can allow arelatively large amount of sample material on a single sample holder(e.g., a substrate) to be imaged and analyzed in a single session. Thiscan increase the throughput of the analysis process compared to anapproach where the substrate is exchanged after analysis of its entiresample material in order to analyze additional sample material on a newsubstrate, which approach may involve intermediate steps of substrateremoval and insertion, sample preparation, and equipment initialization.

In some implementations, differential coupling between, say, first andsecond linear waveguides can include coupling light into the firstlinear waveguide while not coupling any of the light into the secondlinear waveguide, or vice versa. Such differential coupling may notalways be practical or possible. In some implementations, differentialcoupling can involve minimizing the coupling into, say, the secondlinear waveguide while coupling the light into the first linearwaveguide during a portion of the scan. The amount or fraction of theminimization can differ depending on the implementation. In someimplementations, the minimized coupling (e.g., the cross-talk)corresponds to at most about 1%, about 5%, about 15%, about 25% or about45% of the coupling into the linear waveguide. Such differentialcoupling may not always be practical or possible. In someimplementations, differential coupling can involve reducing the couplinginto, say, the second linear waveguide compared to the first linearwaveguide during a portion of the scan. The amount or fraction of thereduction can differ depending on the implementation. In someimplementations, the reduced coupling (e.g., the cross-talk) correspondsto at most about 5%, about 15%, about 35%, about 65% or about 95% of thecoupling into the linear waveguide.

The amount of cross-talk (e.g., the magnitude thereof) may be known orcalibrated. In some implementations, multiple scans of a sample can beperformed, such as a first scan with coupling into the first linearwaveguide where coupling into the second linear waveguide is reduced,and a second scan with coupling into the second linear waveguide wherecoupling into the first linear waveguide is reduced. The scans may causemodulation of the information obtained from the first and second linearwaveguides, respectively. Such modulation may occur in a predictable waygiven the magnitude of the cross-talk. For example, linear algebra canbe applied to the information obtained from the respective first andsecond linear waveguides to extract useful analysis information.

The limit imposed by the maximum available resolution of imagingequipment can be referred to as a diffraction limit. An imaging systemoperating at the maximum resolution thus available can be said to bediffraction-limited. For microscopic instruments the spatial resolutionthat can be obtained given the diffraction limit depends on the lightwavelength and on the numerical aperture of the objective or theillumination source. The minimum resolvable distance d can be expressedas d=λ/(2n sin θ), where λ is the light wavelength, n is the refractiveindex, and θ is the half-angle (i.e., one half of the angle between amicroscope optical axis and the direction of the most oblique light rayscaptured by the objective). The factor n sin θ is usually referred to asthe numerical aperture (NA), and the minimum resolvable distance cantherefore be expressed as d=λ/(2NA). That is, in existing analysissystems the sample material is generally distributed with a density suchthat the individual portions of the sample are at least a distance dapart. Systems and techniques described herein can allow analysis to beperformed on sample material that is distributed more densely than theresolution distance d.

Sample analysis can include, but is not limited to, genetic sequencing(e.g., determining the structure of genetic material), genotyping (e.g.,determining differences in an individual's genetic make-up), geneexpression (e.g., synthesizing a gene product using gene information),proteomics (e.g., large-scale study of proteins), or combinationsthereof.

Some examples described herein relate to sequencing of genetic material.Sequencing can be performed on a sample to determine which buildingblocks, called nucleotides, make up the particular genetic material thatis in the sample. The sequencing can be done after the genetic materialhas first been purified and then replicated a number of times so as toprepare a sample of a suitable size. Imaging can be performed as part ofthe process of sequencing the genetic material. This can involvefluorescent imaging, where a sample of genetic material is subjected tolight (e.g., a laser beam) to trigger a fluorescent response by one ormore markers on the genetic material. Some nucleotides of the geneticmaterial can have fluorescent tags applied to them, which allows fordetermination of the presence of the nucleotide by shining light onto,and looking for a characteristic response from, the sample. Fluorescentresponses can be detected over the course of the sequencing process andused to build a record of nucleotides in the sample.

Examples described herein refer to flowcells. A flowcell can beconsidered a substrate that can be used in preparing and accommodatingor carrying one or more samples in at least one stage of an analysisprocess. The flowcell is made of a material that is compatible with boththe sample material (e.g., genetic material), the illumination and thechemical reactions to which it will be exposed. The substrate can haveone or more channels in which sample material can be deposited. Asubstance (e.g., a liquid) can be flowed through the channel where thesample genetic material is present to trigger one or more chemicalreactions and/or to remove unwanted material. The flowcell may enablethe imaging by facilitating that the sample in the flowcell channel canbe subjected to illuminating light and that any fluorescent responsesfrom the sample can be detected. Some implementations of the system maybe designed to be used with at least one flowcell, but may not includethe flowcell(s) during one or more stages, such as during shipping orwhen delivered to a customer. For example, the flowcell(s) can beinstalled into an implementation at the customer's premises in order toperform analysis.

Examples herein refer to coupling of light (e.g., a laser beam) intoand/or out of a waveguide by one or more gratings. A grating can couplelight impinging on the grating by way of diffracting at least a portionof the light, thereby causing the portion of the light to propagate inone or more other directions. In some implementations, the coupling caninvolve one or more interactions, including, but not limited to,reflection, refraction, diffraction, interference, and/or transmissionof the portion of the light. Implementations may be designed to meet oneor more requirements, including, but not limited to, those regardingmass production, cost control, and/or high light coupling efficiency.Two or more gratings can be identical or similar to each other, ordifferent types of gratings can be used. The grating(s) can include oneor more forms of periodic structure. In some implementations, thegrating can be formed by removing or omitting material from a substrate(e.g., from a waveguide material that is included in the flowcell) orother material. For example, the flowcell can be provided with a set ofslits and/or grooves therein to form the grating. In someimplementations, the grating can be formed by adding matter to theflowcell (e.g., to a waveguide material that is included in theflowcell) or other material. For example, the flowcell can be providedwith a set of ridges, bands or other protruding longitudinal structuresto form the grating. Combinations of these approaches can be used.

Providing a waveguide in a substrate (such as a flowcell) can provideone or more advantages. Excitation using evanescent light based on totalinternal reflection (TIR) can provide a higher efficiency ofillumination. In some previous approaches, the entirety of a laser beamwas used for illuminating the substrate that held the sample, such as ina scanning process. Such an approach may cause a majority of the lightwave simply propagates through the substrate without effectivelyilluminating the sample. As a result, only a small portion of the lightapplied by such systems may actually be used for exciting fluorophoresin the sample. The evanescent light, by contrast, may penetrate material(e.g., a cladding adjacent to the core layer) only to a certain depth(e.g., about 150-200 nm in one example). For example, the flowcell canbe designed with one or more nanowells configured so that the evanescentfield is largely confined to the well area. As a result, evanescentlight may be a very efficient way of exciting fluorophores. For example,a system operating according to an earlier illumination approach mayinvolve a laser with a certain power; using evanescent light, bycontrast, a significantly lower laser power may be sufficient.

Examples herein refer to chemical vapor deposition. Chemical vapordeposition (CVD) may include all techniques where a volatile material(sometimes referred to as a precursor) is caused to undergo reactionand/or decomposition on the surface of a substrate, forming a depositthereon. CVD may be characterized by one or more aspects. For example,CVD may be characterized by the physical characteristic(s) of the vapor(e.g., whether the CVD is aerosol-assisted or involves direct liquidinjection). For example, CVD may be characterized by the type ofsubstrate heating (e.g., whether the substrate is directly heated orindirectly heated, such as by a heated chamber). Examples of types ofCVD that can be used include, but are not limited to, atmosphericpressure CVD, low pressure CVD, very low pressure CVD, ultrahigh vacuumCVD, metalorganic CVD, laser assisted CVD, and plasma-enhanced CVD.

Examples herein refer to atomic layer deposition. Atomic layerdeposition may be considered a form of CVD and include all techniqueswhere a film is grown on a substrate by exposure to gases. For example,gaseous precursors may be alternatingly introduced into a chamber. Themolecules of one of the precursors may react with the surface until alayer is formed and the reaction is terminated, and the next gaseousprecursor may then be introduced to begin forming a new layer, and so onin one or more cycles.

Examples herein refer to spray coating. Spray coating may include any orall techniques by which a particularized material is caused to bedeposited onto a substrate. This may include, but is not limited to,thermal spraying, plasma spraying, cold spraying, warm spraying, and/orother procedures involving atomized or nebulized material.

Examples herein refer to spin coating. Spin coating may includeapplication of an amount of coating material to a substrate, anddistributing or spreading the coating material over the substrate by wayof centrifugal force due to rotation or spinning of the substrate.

Examples herein refer to nanoimprinting. In nanoimprinting lithography,a pre-fabricated nanoscale template may mechanically displace a fluidicresin to mold the desired nanostructures. The resin may then be curedwith the nanoscale template in place. Following the removal of thenanoscale template, a molded solid resin attached to a desired substratemay be produced. In some implementations, a nanoimprinting process maybegin with fully or partially covering a substrate or wafer withimprinting resin (e.g., a resin as exemplified below). One or morenanostructures may be formed in the imprinting resin in a moldingprocess using a nanoscale template. The imprinting resin can be curedagainst the substrate or wafer, and a resin-removal process can beapplied to remove residue from the wafer or substrate. For example, theresin removal can form chamber lanes adjacent to the nanostructures. Thesubstrate or wafer so formed can have another substrate or a gasketapplied thereto so as to form a flowcell having the describednanostructures as well as flowcell chambers formed by enclosing thechamber lanes. In some implementations, the process of applying theimprinting resin may be configured to produce little or no resinresidue, and in such implementations a resin-removal process can beomitted. In some applications, the cured resin may also befunctionalized with a chemical treatment or an attachment ofbiomolecules, depending on the end use. In nanoimprinting lithography,an imprinted photoresist can be a sacrificial material and similarly beused as an intermediate tool to transfer the patterned resist into thesubstrate or a variation of the resist can be used such that theimprinted resist serves as the input to a subsequent coating step. Anexample of a resist that would remain following patterning is materialformed by a process that involves conversion of monomers into acolloidal solution as a precursor to a gel of particles and/or polymers,sometimes referred to as a sol-gel based material.

Examples herein refer to substrates. A substrate may refer to anymaterial that provides an at least substantially rigid structure, or toa structure that retains its shape rather than taking on the shape of avessel to which it is placed in contact. The material can have a surfaceto which another material can be attached including, for example, smoothsupports (e.g., metal, glass, plastic, silicon, and ceramic surfaces),as well as textured and/or porous materials. Possible substratesinclude, but are not limited to, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, plastics, optical fiberbundles, and a variety of other polymers. In general, the substratesallow optical detection and do not themselves appreciably fluoresce.

Examples herein refer to polymers. A polymer layer can include a film ofa polymer material. Example film forming polymers include, withoutlimitation, acrylamide or copolymers with C1-C12; aromatic and hydroxylderivatives; acrylate copolymers; vinylpyrrolidine and vinylpyrrolidonecopolymers; sugar based polymers such as starch or polydextrins; orother polymers such as polyacrylic acid, polyethylene glycol, polylacticacid, silicone, siloxanes, polyethyleneamines, guar gum, carrageenan,alginate, lotus bean gum, methacrylate co polymers, polyimide, a cyclicolefin copolymer, or combinations thereof. In some implementations, apolymer layer comprises at least one photocurable polymer. For example,a photocurable polymer can include urethane, acrylate, silicone, epoxy,polyacrylic acid, polyacrylates, epoxysilicone, epoxy resins,polydimethylsiloxane (PDMS), silsesquioxane, acyloxysilanes, maleatepolyesters, vinyl ethers, monomers with vinyl or ethynyl groups, orcopolymers, or combinations thereof. In some implementations, a layercan include a covalently attached polymer coating. For example, this caninclude a polymer coating that forms chemical bonds with afunctionalized surface of a substrate, as compared to attachment to thesurface in other ways, for example, adhesion or electrostaticinteraction. In some implementations, a polymer comprised in afunctionalizable layer is poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide), sometimes referred to as PAZAM.

Examples described herein mention that one or more resins may be used.Any suitable resin may be used for nanoimprinting in methods describedherein. In some implementations, an organic resin may be used,including, but not limited to, an acrylic resin, a polyimide resin, amelamine resin, a polyester resin, a polycarbonate resin, a phenolresin, an epoxy resin, polyacetal resin, polyether resin, polyurethaneresin, polyamide resin (and/or nylon), a furan resin, a diallylphthalateresin, or combinations thereof. In some examples, a resin may include aninorganic siloxane polymer including a Si—O—Si bond among compounds(including silicon, oxygen, and hydrogen), and formed by using asiloxane polymer-based material typified by silica glass as a startingmaterial. A resin used may also or instead be an organic siloxanepolymer in which hydrogen bonded to silicon is substituted by an organicgroup, such as methyl or phenyl, and typified by an alkylsiloxanepolymer, an alkylsilsesquioxane polymer, a silsesquioxane hydridepolymer, or an alkylsilsesquioxane hydride polymer. Non-limitingexamples of siloxane polymers include polyhedral oligomericsilsesquioxane (POSS), polydimethylsiloxane (PDMS), tetraethyl orthosilicate (TEOS), poly (organo) siloxane (silicone), andperfluoropolyether (PFPE). A resin may be doped with a metal oxide. Insome implementations, a resin may be a sol-gel material including, butnot limited to, titanium oxide, hafnium oxide, zirconium oxide, tinoxide, zinc oxide, or germanium oxide, and that uses a suitable solvent.Any one of a number of other resins may be employed, as appropriate tothe application.

FIG. 1 shows a cross section of part of an example flowcell 100 withlinear waveguides 102A-102C. The flowcell 100 can be used with one ormore methods described herein, and/or be used in combination with one ormore systems or apparatuses described herein. Only a portion of theflowcell 100 is shown, for purposes of illustration. For example, one ormore additional layers and/or more or fewer waveguides 102A-102C, can beused.

The flowcell 100 includes a substrate 104. The substrate 104 can form abase for the flowcell 100. In some implementations, one or more otherlayers can be formed at (e.g., in contact with or near) the substrate104 in the manufacturing of the flowcell 100. The substrate 104 canserve as a basis for forming the linear waveguides 102A-102C. The linearwaveguides 102A-102C can initially exist separately from the substrate104 and thereafter be applied onto the substrate 104, or the linearwaveguides 102A-102C can be formed by application, and/or removal of,one or more materials to or from the substrate. The linear waveguides102A-102C can be formed directly onto the substrate 104, or onto one ormore intermediate layers at the substrate 104.

The linear waveguides 102A-102C serve to conduct electromagneticradiation (including, but not limited to, visible light, such as laserlight). In some implementations, the electromagnetic radiation performsone or more functions during an imaging process. For example, theelectromagnetic radiation can serve to excite fluorophores in a samplematerial for imaging. The linear waveguides 102A-102C can be made of anysuitable material that facilitates propagation of one or more kinds ofelectromagnetic radiation. In some implementations, the material(s) ofthe linear waveguides 102A-102C can include a polymer material. In someimplementations, the material(s) of the linear waveguides 102A-102C caninclude Ta₂O₅ and/or SiN_(x). For example, the linear waveguides102A-102C can be formed by sputtering, chemical vapor deposition, atomiclayer deposition, spin coating, and/or spray coating.

Each of the linear waveguides 102A-102C can have one or more gratings(omitted here for clarity) to couple electromagnetic radiation intoand/or out of that linear waveguide 102A-102C. One or more directions oftravel for the electromagnetic radiation in the linear waveguides102A-102C can be employed. For example, the direction of travel can beinto and/or out of the plane of the present illustration. Examples ofgratings are described elsewhere herein.

Each of the linear waveguides 102A-102C can be positioned against one ormore types of cladding. The cladding can serve to constrain theelectromagnetic radiation to the respective linear waveguide 102A-102Cand prevent, or reduce the extent of, propagation of the radiation intoother linear waveguides 102A-102C or other substrates. Here, claddings106A-106D are shown as an example. For example, the claddings 106A-106Bcan be positioned against or near the linear waveguide 102A on different(e.g., opposing) sides thereof. For example, the claddings 106B-106C canbe positioned against or near the linear waveguide 102B on different(e.g., opposing) sides thereof. For example, the claddings 106C-106D canbe positioned against or near the linear waveguide 102C on different(e.g., opposing) sides thereof. The claddings 106A-106D can be made fromone or more suitable materials that serve to separate the linearwaveguides 102A-102C from each other. In some implementations, thecladdings 106A-106D can be made from a material having a lowerrefractive index than the refractive index/indices of the linearwaveguides 102A-102C. For example, the linear waveguides 102A-102C canhave a refractive index of about 1.4-1.6, and the claddings 106A-106Dcan have a refractive index of about 1.2-1.4. In some implementations,one or more of the claddings 106A-106D includes a polymer material. Insome implementations, one or more of the claddings 106A-106D includesmultiple structures, including, but not limited to, structures of onematerial (e.g., polymer) interspersed by regions of vacuum or anothermaterial (e.g., air or a liquid).

The flowcell 100 includes at least one nanowell layer 108. In someimplementations, the nanowell layer 108 is positioned opposite thelinear waveguides 102A-102C from the substrate 104. For example, thenanowell layer can be positioned adjacent (e.g., abutting or near) thelinear waveguides 102A-102C and the claddings 106A-106D. The nanowelllayer 108 includes one or more nanowells. In some implementations, thenanowell layer 108 includes nanowells 108A-108C. The nanowells 108A-108Ccan be used for holding one or more sample materials during at leastpart of the analysis process (e.g., for imaging). For example, one ormore genetic materials (e.g., in form of clusters) can be placed in thenanowells 108A-108C.

One or more of the nanowells 108A-108C can be at least substantiallyaligned with one or more of the linear waveguides 102A-102C. This canallow interaction between the respective nanowell 108A-108C and thecorresponding linear waveguide 102A-102C for imaging purposes(including, but not limited to, by way of transmission of evanescentlight). For example, the nanowell 108A can be at least substantiallyaligned with the linear waveguide 102A; the nanowell 108B can be atleast substantially aligned with the linear waveguide 102B; and/or thenanowell 108C can be at least substantially aligned with the linearwaveguide 102C.

The nanowells 108A-108C can be formed by nanoimprinting into thenanowell layer 108, or a lift-off process from the nanowell layer 108.For example, the nanowell layer 108 can include a resin and thenanowells 108A-108C can be formed by imprinting using a nanoscaletemplate. In some implementations, the nanowells 108A-108C can have asize such that one or more of its dimensions ranges in the order of oneor more nanometers. An end (e.g., the bottom) of the nanowells 108A-108Ccan have a thickness that accommodates propagation of evanescent light.For example, the thickness can be about 0-500 nm. The nanowell layer cancover at least substantially the entire facing surface of the layer thatincludes the linear waveguides 102A-102C and the claddings 106A-106D. Insome implementations, the nanowell layer 108 can have an average pitchbetween the nanowells 108A-108C of at least 10 nm, 0.1 μm, 0.5 μm, 1 μm,5 μm, 10 μm, 100 μm or more, and/or can have an average pitch of at most100 μm, 10 μm, 5 μm, 0.5 μm 0.1 μm or less. In some implementations, thenanowell layer 108 can have a pitch between the nanowells 108A-108C ofabout 150 nm or greater. For example, the nanowell layer 108 can have apitch between the nanowells 108A-108C of about 160 nm, 220 nm, 250 nm,300 nm, 450 nm, or greater. The depth of each nanowell 108A-108C can beat least 0.1 μm, 10 μm, 100 μm or more. Alternatively or additionally,the depth can be at most 1×10³ μm, 100 μm, 10 μm, 0.1 μm or less.

FIGS. 2A-2B illustrate examples with a flowcell 200 having staggeredgratings 202. The flowcell 200 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. Only a portion of the flowcell 200 isshown, for purposes of illustration.

The flowcell 200 includes nanowells, including a nanowell 204A, that arehere illustrated using circular shapes. Only some of the nanowells willbe specifically mentioned, and the other nanowells may be similar oridentical to the one(s) discussed. The nanowells may be formed in ananowell layer (e.g., by way of nanoimprinting or a lift-off process).For example, the nanowells can be formed in a resin using a nanoscaletemplate. The nanowell layer is not explicitly shown in this example,for purposes of clarity. The nanowell 204A is here associated with alinear waveguide 206A. In some implementations, the linear waveguidesdescribed with reference to the flowcell 200 can be similar or identicalto one or more other linear waveguides described herein. For example,the linear waveguide 206A is positioned adjacent (e.g., in contact withor near) the nanowell layer that includes the nanowell 204A. In someimplementations, the linear waveguide 206A can include a linearwaveguide core 208 and one or more of the gratings 202.

Another nanowell 204B is also associated with the linear waveguide 206A.For example, the nanowell 204B is positioned adjacent to the nanowell204A and both of the nanowells 204A-204B can interact with the linearwaveguide 206A in an imaging process (e.g., by way of receivingelectromagnetic radiation from the linear waveguide 206A). Anothernanowell 204C, by contrast, is instead associated with a linearwaveguide 206B. In some implementations, the linear waveguide 206B ispositioned adjacent to the linear waveguide 206A. For example, cladding(not shown) and/or another material can be positioned between the linearwaveguides 206A-206B.

Some examples described herein mention or otherwise relate to sets ofnanowells. A set of nanowells is a logical or physical group of one ormore nanowells having at least one characteristic. A set of nanowellsmay be associated with one linear waveguide, and another set ofnanowells may be associated with another linear waveguide. In someimplementations, a set of nanowells may be arranged in a row. Such a rowof nanowells can extend along the linear waveguide, such as by beingcoextensive with (e.g., fully overlapping above or below) the linearwaveguide, or by being parallel to and positioned adjacent (e.g., oneither or both sides of) the linear waveguide, to name just someexamples. Accordingly, a set of nanowells can include one or more rowsof nanowells in some implementations. Each of such rows of nanowells canbe aligned with at least one linear waveguide.

Nanowells can be arranged on a substrate (e.g., in a nanowell layer) ina substantially, and in at least one instance completely, random way oraccording to one or more patterns. In some implementations, thenanowells are arranged in form of one or more arrays, including, but notlimited to, a polygonal array. For example, a polygonal array can be arectangular, triangular, or a hexagonal array, or any other form ofarray where at least some nanowells are arranged in a polygon shape. Theflowcell 200 in this example has a rectangular array of nanowells.

The flowcell 200 can be used in one or more forms of imaging process.For example, sample material in the nanowells (including the nanowells204A-204C) can be subjected to electromagnetic radiation from respectivelinear waveguides (including the linear waveguides 206A-206B,respectively). Emissions resulting from such exposure to electromagneticradiation (an example of emissions being fluorescence from fluorophores)can be captured using equipment (e.g., one or more cameras and/or otherimaging devices). Such equipment is sometimes referred to by way of theexpression emission equipment or a similar term. For example, emissionequipment can include one or more cameras or other image sensors and atleast one lens or other emission optics. In some implementations, thediffraction limit can be at least partially attributable to one or morecharacteristics of the emission optics. For example, based on theemission optics used, a resolution distance can be defined, theresolution distance marking the shortest distance that can be resolvedusing the emission optics. That is, when resolving features that arespaced apart by the resolution distance, the imaging system can be saidto be operating at its highest available level of resolution.

Here, a distance 210 is less than the resolution distance of theemission optics, and a distance 212 is greater than, or about equal to,the resolution distance of the emission optics. The distance 210 hererepresents the separation between nanowells in one direction. In someimplementations, this can be the direction across the linear waveguides.For example, because the linear waveguides are here aligned with rows ofthe nanowells in one direction (e.g., the vertical direction as seen inthe illustration), the distance 210 can also represent the distancebetween adjacent linear waveguides (e.g., the linear waveguides206A-206B). For example, the nanowells 204A and 204C are separated bythe distance 210. That is, the linear waveguides 206A-206B arepositioned closer to each other than the resolution distance of theemission optics.

The distance 212 here represents the separation between nanowells inanother direction than the distance 210. For example, the distances 210and 212 can be substantially, and in at least one instance completely,perpendicular to each other. In some implementations, this can be thedirection along any individual one of the linear waveguides. Forexample, because the linear waveguides are here aligned with rows of thenanowells in one direction (e.g., the vertical direction as seen in theillustration), the distance 212 can represent the distance betweenadjacent nanowells on any of the linear waveguides (e.g., the linearwaveguides 206A-206B). For example, the nanowells 204A and 204B areseparated by the distance 212. That is, the nanowells associated withthe linear waveguide 206A have a spacing from each other that isresolvable according to the resolution distance of emission optics forthe flowcell 200.

The gratings 202 serve for coupling electromagnetic radiation intoand/or out of the linear waveguides of the flowcell 200. Here, thelinear waveguide 206A has a grating 202A and the linear waveguide 206Bhas a grating 202B. The gratings 202A-202B can have the same ordifferent periodic structure. In some implementations, either or both ofthe gratings 202A-202B can include a periodic structure of ridgesinterspersed by another material. For example, ridges of the gratings202A-202B can have a pitch of about 200-300 nm, to name just oneexample.

The gratings 202A-202B can have one or more characteristics thatfacilitate selective coupling of electromagnetic radiation into thecorresponding linear waveguide 206A-206B. In some implementations, oneor more of the gratings 202 is spatially offset from one or more othersof the gratings 202. The offset can be in a direction that is parallelto the linear waveguides 206A-206B. For example, the distance betweenthe grating 202B and the closest nanowell of the nanowells associatedwith the linear waveguide 206B is here greater than the distance betweenthe grating 202A and the closest nanowell of the nanowells associatedwith the linear waveguide 206A. The characteristic of the gratings202A-202B being spatially offset from each other facilitates coupling ofelectromagnetic radiation (e.g., light) into one of the linearwaveguides (e.g., the linear waveguide 206A) without coupling theelectromagnetic radiation (e.g., light) into another of the linearwaveguides (e.g., the linear waveguide 206B).

The flowcell 200 can include multiple linear waveguides, for example asillustrated. In some implementations, a linear waveguide 206C ispositioned adjacent to the linear waveguide 206B opposite from thelinear waveguide 206A. For example, the linear waveguide 206C can have agrating 202C. In some implementations, the grating 202C can be spatiallyoffset from the grating 202B. For example, the grating 202C can have thesame spatial offset from the grating 202B, in the direction parallel tothe linear waveguide 206C, as the grating 202A has in the directionparallel to the linear waveguide 206A.

The characteristic of the gratings 202A and 202C being spatially offsetfrom the grating 202B facilitates coupling of electromagnetic radiation(e.g., light) into one of the linear waveguides (e.g., the linearwaveguide 206A or 206C) without coupling the electromagnetic radiation(e.g., light) into another of the linear waveguides (e.g., the linearwaveguide 206B). As another example, the characteristic facilitatescoupling of electromagnetic radiation (e.g., light) into one of thelinear waveguides (e.g., the linear waveguide 206B) without coupling theelectromagnetic radiation (e.g., light) into at least one other of thelinear waveguides (e.g., the linear waveguide 206A or 206C).

A light area 214 is here schematically illustrated as a rectangle with adashed outline. The light area 214 represents one or more positionswhere light or other electromagnetic radiation is caused to impinge aspart of an imaging process. In some implementations, illuminating lightgenerated by a laser can be directed at the light area 214 in order toeventually be coupled into some of the linear waveguides. For example,the laser light can be selected so as to correspond to fluorescenceproperties of one or more fluorophores in the sample material.

An image capture area 216 is here schematically illustrated as arectangle with a dashed outline. The image capture area 216 representsthe field of view of the emission optics. For example, a camera or otherimage sensor can capture one or more types of emissions (e.g.,fluorescent light) emanating from the image capture area 216.

The examples described above illustrate that the flowcell 200 includes ananowell layer having first (e.g., the nanowells associated with thelinear waveguide 206A) and second (e.g., the nanowells associated withthe linear waveguide 206B) sets of nanowells to receive a sample. Theflowcell 200 includes a first linear waveguide (e.g., the linearwaveguide 206A) aligned with the first set of nanowells, and a secondlinear waveguide (e.g., the linear waveguide 206B) aligned with thesecond set of nanowells; and a first grating (e.g., the grating 202A)for the first linear waveguide, and a second grating (e.g., the grating202B) for the second linear waveguide. The first grating has a firstcharacteristic (e.g., being spatially offset from the grating 202B) tofacilitate coupling of first light into the first linear waveguidewithout coupling the first light into the second linear waveguide.

An image capture process can include one or more scanning operations. Insome implementations, the image capture area 216 can be caused tooverlay one or more areas of the flowcell 200 to facilitate imagecapture regarding one or more nanowells in the image capture area 216.The positioning can include movement of the image capture area 216, orthe flowcell 200, or both. For example, the emission optics can berelatively stationary in the analysis equipment, such that the imagecapture area 216 does not move during various scanning operations. Forexample, the flowcell 200 can be moved (e.g., by being positioned on amotorized stage that facilitates precise movement in at least onedirection) relative to the image capture area 216 into one or morescanning positions. Here, an arrow 218 schematically illustrates thatthe flowcell 200 can be moved so that the image capture area 216overlays at least some of the linear waveguides and the nanowellsassociated with them.

The light area 214 can remain stationary with, or be moved correspondingto, or be moved independently of, the image capture area 216. In thisexample, the light area 214 is aligned with some of the gratings 202(e.g., with the gratings 202A and 202C) but is not aligned with someother ones of the gratings (e.g., with the grating 202B). For example,when scanning is done in the direction of the arrow 218 with the currentposition of the light area 214, the gratings 202A and 202C (and othershaving similar spatial offset) will be illuminated by the lightimpinging at the light area 214, whereas some other ones of the gratings(e.g., the grating 202B) will not be illuminated by the light impingingat the light area 214. Accordingly, illuminating light will be coupledinto the linear waveguides 206A and 206C (and others whose gratings havesimilar spatial offsets), whereas the light will not be coupled into thelinear waveguide 206B (and others whose gratings have similar spatialoffsets). This can facilitate a selective illumination of the nanowellsof the flowcell 200. For example, because the linear waveguides 206A and206C have light coupled into them, excitation light can reach thenanowells 204A and 204C associated with the linear waveguide 206A, and ananowell 204D associated with the linear waveguide 206C. On the otherhand, excitation light should not reach the nanowell 204C because it isassociated with the linear waveguide 206B which does not currently havelight coupled into it. As such, the imaging can successfully proceedalthough some portions of the sample material (e.g., within thenanowells 204A and 204C) are positioned at the distance 210 from eachother; that is, closer to each other than the resolution distance of theemission optics. During the scanning corresponding to the movementrepresented by the arrow 218 (which may be characterized as a linescan), only a particular subset of the linear waveguides can have lightcoupled into them. In some implementations, light is coupled into onlyevery second linear waveguide. For example, light may be coupled intoonly the first, third, fifth, seventh, and so on, linear waveguide,whereas light is not coupled into the second, fourth, sixth, eighth, andso on, linear waveguide.

In some implementations, the distance 210 is shorter than a diffractionlimit (e.g., a resolution distance of the emission optics). For example,if the wavelength is about 700 nm with a 0.75 numerical aperture, thediffraction limit is about 466 nm, and the distance 210 can then beshorter than this limit. In some implementations, the flowcell 200 canbe designed so that the nanowells 204A and 204D are separated from eachother by about the diffraction limit (e.g., by about 466 nm). Forexample, the distance 210 can then be about half of the diffractionlimit (e.g., about 233 nm). As another example, if the wavelength isabout 525 nm with a 0.75 numerical aperture, the diffraction limit isabout 350 nm, and the distance 210 can then be about 175 nm. The aboveexample involves activating every other linear waveguide at a time. Insome implementations, fewer than every other linear waveguide can beactuated at a time. For example, if every third linear waveguide isactivated at a time, then the distance 210 can be about one third of thediffraction limit. As another example, if every fourth linear waveguideis activated at a time, then the distance 210 can be about one fourth ofthe diffraction limit, and so on.

The scanning being illustrated in FIG. 2A can be described as theflowcell 200 being moved to the left in the image, and stopping at oneor more selected positions corresponding to the linear waveguides as theimage capture area 216 overlays them, until the flowcell 200 is to theleft of the image capture area 216. One or more linear waveguides whichdo not have light coupled into them during the scan illustrated in FIG.2A, and whose associated nanowells are accordingly not then subjected toexcitation light, can be imaged in another scanning operation.

Such other scanning operation can be performed in the same direction asdescribed above (e.g., along the direction of the arrow 218) or inanother direction. FIG. 2B shows an example where scanning is done alonga direction corresponding to an arrow 220, which direction issubstantially, and in at least one instance completely, opposite to thedirection associated with the arrow 218. The scanning being illustratedin FIG. 2B can be described as the flowcell 200 being moved to the rightin the image, and stopping at one or more selected positionscorresponding to the linear waveguides as the image capture area 216overlays them, until the flowcell 200 is to the right of the imagecapture area 216. The positioning can include movement of the imagecapture area 216, or the flowcell 200, or both.

In this example, the light area 214 is aligned with some of the gratings202 (e.g., with the grating 202B) but is not aligned with some otherones of the gratings (e.g., with the gratings 202A and 202C). Forexample, when scanning is done in the direction of the arrow 220 withthe current position of the light area 214, the grating 202B (and othershaving similar spatial offset) will be illuminated by the lightimpinging at the light area 214, whereas some other ones of the gratings(e.g., the gratings 202A and 202C) will not be illuminated by the lightimpinging at the light area 214. Accordingly, illuminating light will becoupled into the linear waveguide 206B (and others whose gratings havesimilar spatial offsets), whereas the light will not be coupled into thelinear waveguides 206A and 206C (and others whose gratings have similarspatial offsets). This can facilitate a selective illumination of thenanowells of the flowcell 200. For example, because the linear waveguide206C has light coupled into it, excitation light can reach the nanowell204C and others associated with the linear waveguide 206B. On the otherhand, excitation light should not reach the nanowells 204A-204B that areassociated with the linear waveguide 206A, or the nanowell 204D that isassociated with the linear waveguide 206C, which linear waveguides donot currently have light coupled into them. As such, the imaging cansuccessfully proceed although some portions of the sample material(e.g., within the nanowells 204A and 204C) are positioned at thedistance 210 from each other; that is, closer to each other than theresolution distance of the emission optics. During the scanningcorresponding to the movement represented by the arrow 220 (which may becharacterized as a line scan), only a particular subset of the linearwaveguides can have light coupled into them. In some implementations,light is coupled into only every second linear waveguide. For example,light may be coupled into only the second, fourth, sixth, eighth, and soon, linear waveguide, whereas light is not coupled into the first,third, fifth, seventh, and so on, linear waveguide.

The examples relating to FIGS. 2A-2B relate to differential couplingwhere the gratings 202 are spatially offset from each other. In someimplementations, one or more other approaches can instead or also beused for differential coupling. Such approaches can include, but are notlimited to, differentiated beam parameters, differentiated couplerparameters, and/or differentiated waveguide parameters. Examples areprovided below.

FIGS. 3A-3B illustrate examples with a flowcell 300 having gratings 302.In some implementations, differential coupling for the flowcell 300 isprovided based on differentiating one or more parameters of the lightbeam(s) applied to the gratings 302. In some implementations,differential coupling for the flowcell 300 is provided based ondifferentiating one or more parameters of the gratings 302. In someimplementations, differential coupling for the flowcell 300 is providedbased on differentiating one or more parameters of the linear waveguidesof the flowcell 300. Combinations of two or more of these approaches canbe used. The flowcell 300 can be used with one or more methods describedherein, and/or be used in combination with one or more systems orapparatuses described herein. Only a portion of the flowcell 300 isshown, for purposes of illustration.

The flowcell 300 includes nanowells, including a nanowell 304A, that arehere illustrated using circular shapes. Only some of the nanowells willbe specifically mentioned, and the other nanowells may be similar oridentical to the one(s) discussed. The nanowells may be formed in ananowell layer (e.g., by way of nanoimprinting or a lift-off process).For example, the nanowells can be formed in a resin using a nanoscaletemplate. The nanowell layer is not explicitly shown in this example,for purposes of clarity. The nanowell 304A is here associated with alinear waveguide 306A. In some implementations, the linear waveguidesdescribed with reference to the flowcell 300 can be similar or identicalto one or more other linear waveguides described herein. For example,the linear waveguide 306A is positioned adjacent (e.g., in contact withor near) the nanowell layer that includes the nanowell 304A. In someimplementations, the linear waveguide 306A can include a linearwaveguide core 308 and one or more of the gratings 302.

Another nanowell 304B is also associated with the linear waveguide3206A. For example, the nanowell 304B is positioned adjacent to thenanowell 304A and both of the nanowells 304A-304B can interact with thelinear waveguide 306A in an imaging process (e.g., by way of receivingelectromagnetic radiation from the linear waveguide 306A). Anothernanowell 304C, by contrast, is instead associated with a linearwaveguide 306B. In some implementations, the linear waveguide 306B ispositioned adjacent to the linear waveguide 306A. For example, cladding(not shown) and/or another material can be positioned between the linearwaveguides 306A-306B.

The flowcell 300 can be used in one or more forms of imaging process.For example, sample material in the nanowells (including the nanowells304A-304C) can be subjected to electromagnetic radiation from respectivelinear waveguides (including the linear waveguides 306A-306B,respectively). Emissions resulting from such exposure to electromagneticradiation (an example of emissions being fluorescence from fluorophores)can be captured using equipment (e.g., one or more cameras and/or otherimaging devices). Such equipment is sometimes referred to as emissionequipment or a similar term. For example, emission equipment can includeone or more cameras or other image sensors and at least one lens orother emission optics. In some implementations, the diffraction limitcan be at least partially attributable to one or more characteristics ofthe emission optics. For example, based on the emission optics used, aresolution distance can be defined, the resolution distance marking theshortest distance that can be resolved using the emission optics. Thatis, when resolving features that are spaced apart by the resolutiondistance, the imaging system can be said to be operating at its highestavailable level of resolution.

Here, a distance 310 is less than the resolution distance of theemission optics, and a distance 312 is greater than, or about equal to,the resolution distance of the emission optics. The distance 310 hererepresents the separation between nanowells in one direction. In someimplementations, this can be the direction across the linear waveguides.For example, because the linear waveguides are here aligned with rows ofthe nanowells in one direction (e.g., the vertical direction as seen inthe illustration), the distance 310 can also represent the distancebetween adjacent linear waveguides (e.g., the linear waveguides306A-306B). For example, the nanowells 304A and 304C are separated bythe distance 310. That is, the linear waveguides 306A-306B arepositioned closer to each other than the resolution distance of theemission optics.

The distance 312 here represents the separation between nanowells inanother direction than the distance 310. For example, the distances 310and 312 can be substantially, and in at least one instance completely,perpendicular to each other. In some implementations, this can be thedirection along any individual one of the linear waveguides. Forexample, because the linear waveguides are here aligned with rows of thenanowells in one direction (e.g., the vertical direction as seen in theillustration), the distance 312 can represent the distance betweenadjacent nanowells on any of the linear waveguides (e.g., the linearwaveguides 306A-306B). For example, the nanowells 304A and 304B areseparated by the distance 312. That is, the nanowells associated withthe linear waveguide 306A have a spacing from each other that isresolvable according to the resolution distance of emission optics forthe flowcell 300.

The gratings 302 serve for coupling electromagnetic radiation intoand/or out of the linear waveguides of the flowcell 300. Here, thelinear waveguide 306A has a grating 302A and the linear waveguide 306Bhas a grating 302B. The gratings 302A-302B here have different periodicstructures. In some implementations, either or both of the gratings302A-302B can include a periodic structure of ridges interspersed byanother material. For example, ridges of the gratings 302A-302B can havea pitch of about 200-300 nm, to name just one example.

The gratings 302A-302B can have one or more characteristics thatfacilitate selective coupling of electromagnetic radiation into thecorresponding linear waveguide 306A-306B. In some implementations, oneor more of the gratings 302 has a different grating period from one ormore others of the gratings 302. For example, the grating 302A may havea higher grating period than the grating 302B. As another example, thegrating 302B may have a higher grating period than the grating 302A. Thecharacteristic of the gratings 302A-302B having different gratingperiods from each other facilitates coupling of electromagneticradiation (e.g., light) into one of the linear waveguides (e.g., thelinear waveguide 306A) without coupling the electromagnetic radiation(e.g., light) into another of the linear waveguides (e.g., the linearwaveguide 306B).

In some implementations, coupling into the gratings 302A-302C can alsoor instead be differentiated by a coupler parameter other than gratingperiod (e.g., but not limited to, refractive index, pitch, groove width,groove height, groove spacing, grating non-uniformity, grooveorientation, groove curvature, overall shape of the coupler, andcombinations thereof). In some implementations, coupling into thegratings 302A-302C can also or instead be differentiated by a waveguideparameter regarding one or more linear waveguides of the flowcell 300(e.g., but not limited to, cross-sectional profile, refractive indexdifference, mode matching with the coupler and/or beam, and combinationsthereof). In some implementations, coupling into the gratings 302A-302Ccan also or instead be differentiated by a beam parameter of the lightbeam(s) applied to the flowcell 300 (e.g., but not limited to, locationof the light beam, angle of incidence, divergence, mode profile,polarization, aspect ratio, diameter, wavelength, and combinationsthereof).

The flowcell 300 can include multiple linear waveguides, for example asillustrated. In some implementations, a linear waveguide 306C ispositioned adjacent to the linear waveguide 306B opposite from thelinear waveguide 306A. In other words, in this implementation, thelinear waveguide 306B is between the linear waveguide 306A and linearwaveguide 306C. For example, the linear waveguide 306C can have agrating 302C. In some implementations, the grating 302C can have adifferent grating period from the grating 302B. For example, the grating302C can have the same grating period as the grating 302A. As anotherexample, the grating 302C can have a different grating period from thegrating 302A and from the grating 302B. The characteristic of at leastsome of the gratings 302A-302C having different grating periodsfacilitates coupling of electromagnetic radiation (e.g., light) into oneof the linear waveguides (e.g., the linear waveguide 306A or 306C)without coupling the electromagnetic radiation (e.g., light) intoanother of the linear waveguides (e.g., the linear waveguide 306B). Asanother example, the characteristic facilitates coupling ofelectromagnetic radiation (e.g., light) into one of the linearwaveguides (e.g., the linear waveguide 306B) without coupling theelectromagnetic radiation (e.g., light) into at least one other of thelinear waveguides (e.g., the linear waveguide 306A or 306C).

A light area 314 is here schematically illustrated as a rectangle with adashed outline. The light area 314 represents one or more positionswhere light or other electromagnetic radiation is caused to impinge aspart of an imaging process. In some implementations, illuminating lightgenerated by a laser can be directed at the light area 314 in order toeventually be coupled into some of the linear waveguides. For example,the laser light can be selected so as to correspond to fluorescenceproperties of one or more fluorophores in the sample material.

An image capture area 216 is here schematically illustrated as arectangle with a dashed outline. The image capture area 316 representsthe field of view of the emission optics. For example, a camera or otherimage sensor can capture one or more types of emissions (e.g.,fluorescent light) emanating from the image capture area 316.

The examples described above illustrate that the flowcell 300 includes ananowell layer having first (e.g., the nanowells associated with thelinear waveguide 306A) and second (e.g., the nanowells associated withthe linear waveguide 306B) sets of nanowells to receive a sample. Theflowcell 300 includes a first linear waveguide (e.g., the linearwaveguide 306A) aligned with the first set of nanowells, and a secondlinear waveguide (e.g., the linear waveguide 306B) aligned with thesecond set of nanowells; and a first grating (e.g., the grating 302A)for the first linear waveguide, and a second grating (e.g., the grating302B) for the second linear waveguide. The first grating has a firstcharacteristic (e.g., having a different grating period from the grating302B) to facilitate coupling of first light into the first linearwaveguide without coupling the first light into the second linearwaveguide.

An image capture process can include one or more scanning operations. Insome implementations, the image capture area 316 can be caused tooverlay one or more areas of the flowcell 300 to facilitate imagecapture regarding one or more nanowells in the image capture area 316.The positioning can include movement of the image capture area 316, orthe flowcell 300, or both. For example, the emission optics can berelatively stationary in the analysis equipment, such that the imagecapture area 316 does not move during various scanning operations. Forexample, the flowcell 300 can be moved (e.g., by being positioned on amotorized stage that facilitates precise movement in at least onedirection) relative to the image capture area 316 into one or morescanning positions. Here, an arrow 318 schematically illustrates thatthe flowcell 300 can be moved so that the image capture area 316overlays at least some of the linear waveguides and the nanowellsassociated with them.

The light area 314 can remain stationary with, or be moved correspondingto, or be moved independently of, the image capture area 316. In thisexample, the light area 314 is aligned with all of the gratings 302 ofthe flowcell 300. Different incidence angles can be given to theilluminating light that impinges on the light area 314, in order toselectively couple light into at least one, but not at least one other,of the linear waveguides of the flowcell 300. For example, when scanningis done in the direction of the arrow 318, the incident angle can bechosen such that the gratings 302A and 302C (and others having similargrating periods) will couple the light impinging at the light area 314,whereas some other ones of the gratings (e.g., the grating 302B) willnot couple the light impinging at the light area 314. Accordingly,illuminating light will be coupled into the linear waveguides 306A and306C (and others whose gratings have similar grating periods), whereasthe light will not be coupled into the linear waveguide 306B (and otherswhose gratings have similar grating periods). This can facilitate aselective illumination of the nanowells of the flowcell 300. Forexample, because the linear waveguides 306A and 306C have light coupledinto them, excitation light can reach the nanowells 304A and 304Cassociated with the linear waveguide 306A, and a nanowell 304Dassociated with the linear waveguide 306C. On the other hand, excitationlight should not reach the nanowell 304C because it is associated withthe linear waveguide 306B which does not currently have light coupledinto it. As such, the imaging can successfully proceed although someportions of the sample material (e.g., within the nanowells 304A and304C) are positioned at the distance 310 from each other; that is,closer to each other than the resolution distance of the emissionoptics. During the scanning corresponding to the movement represented bythe arrow 318 (which may be characterized as a line scan), only aparticular subset of the linear waveguides can have light coupled intothem. In some implementations, light is coupled into only every secondlinear waveguide. For example, light may be coupled into only the first,third, fifth, seventh, and so on, linear waveguide, whereas light is notcoupled into the second, fourth, sixth, eighth, and so on, linearwaveguide.

The scanning being illustrated in FIG. 3A can be described as theflowcell 300 being moved to the left in the image, and stopping at oneor more selected positions corresponding to the linear waveguides as theimage capture area 316 overlays them, until the flowcell 300 is to theleft of the image capture area 316. One or more linear waveguides whichdo not have light coupled into them during the scan illustrated in FIG.3A, and whose associated nanowells are accordingly not then subjected toexcitation light, can be imaged in another scanning operation.

Such other scanning operation can be performed in the same direction asdescribed above (e.g., along the direction of the arrow 318) or inanother direction. FIG. 3B shows an example where scanning is done alonga direction corresponding to an arrow 320, which direction issubstantially, and in at least one instance completely, opposite to thedirection associated with the arrow 318. The scanning being illustratedin FIG. 3B can be described as the flowcell 300 being moved to the rightin the image, and stopping at one or more selected positionscorresponding to the linear waveguides as the image capture area 316overlays them, until the flowcell 300 is to the right of the imagecapture area 316. The positioning can include movement of the imagecapture area 316, or the flowcell 300, or both.

In this example, the light area 314 is aligned with all of the gratings302 of the flowcell 300. Different incidence angles can be given to theilluminating light that impinges on the light area 314, in order toselectively couple light into at least one, but not at least one other,of the linear waveguides of the flowcell 300. For example, when scanningis done in the direction of the arrow 320, the incident angle can bechosen such that the grating 302B (and others having similar gratingperiods) will couple the light impinging at the light area 314, whereassome other ones of the gratings (e.g., the gratings 302A and 302C) willnot couple the light impinging at the light area 314. Accordingly,illuminating light will be coupled into the linear waveguide 306B (andothers whose gratings have similar grating periods), whereas the lightwill not be coupled into the linear waveguides 306A and 306C (and otherswhose gratings have similar grating periods). This can facilitate aselective illumination of the nanowells of the flowcell 300. Forexample, because the linear waveguide 306B has light coupled into it,excitation light can reach the nanowell 304C and others associated withthe linear waveguide 306B. On the other hand, excitation light shouldnot reach the nanowells 304A-304B that are associated with the linearwaveguide 306A, or the nanowell 304D that is associated with the linearwaveguide 306C, which linear waveguides do not currently have lightcoupled into them. As such, the imaging can successfully proceedalthough some portions of the sample material (e.g., within thenanowells 304A and 304C) are positioned at the distance 310 from eachother; that is, closer to each other than the resolution distance of theemission optics. During the scanning corresponding to the movementrepresented by the arrow 320 (which may be characterized as a linescan), only a particular subset of the linear waveguides can have lightcoupled into them. In some implementations, light is coupled into onlyevery second linear waveguide. For example, light may be coupled intoonly the second, fourth, sixth, eighth, and so on, linear waveguide,whereas light is not coupled into the first, third, fifth, seventh, andso on, linear waveguide.

In some implementations, two or more of the gratings 302 can instead oralso have different refractive indices. For example, this can allowdifferential coupling regarding at least some of the linear waveguides306A-306C.

FIG. 4 shows another example of a flowcell 400 having staggered gratings402. The flowcell 400 can be used with one or more methods describedherein, and/or be used in combination with one or more systems orapparatuses described herein. Only a portion of the flowcell 400 isshown, for purposes of illustration.

The flowcell 400 includes nanowells, including a nanowell 404A, that arehere illustrated using circular shapes. Only some of the nanowells willbe specifically mentioned, and the other nanowells may be similar oridentical to the one(s) discussed. The nanowells may be formed in ananowell layer (e.g., by way of nanoimprinting or a lift-off process).For example, the nanowells can be formed in a resin using a nanoscaletemplate. The nanowell layer is not explicitly shown in this example,for purposes of clarity. The nanowell 404A is here associated with alinear waveguide 406A. In some implementations, the linear waveguidesdescribed with reference to the flowcell 4200 can be similar oridentical to one or more other linear waveguides described herein. Forexample, the linear waveguide 406A is positioned adjacent (e.g., incontact with or near) the nanowell layer that includes the nanowell404A.

Another nanowell 404B is also associated with the linear waveguide 406A.For example, the nanowell 404B is positioned adjacent to the nanowell404A and both of the nanowells 404A-404B can interact with the linearwaveguide 406A in an imaging process (e.g., by way of receivingelectromagnetic radiation from the linear waveguide 406A). Anothernanowell 404C, by contrast, is instead associated with a linearwaveguide 406B. In some implementations, the linear waveguide 406B ispositioned adjacent to the linear waveguide 406A. For example, cladding(not shown) and/or another material can be positioned between the linearwaveguides 406A-406B.

The flowcell 400 can be used in one or more forms of imaging process.For example, sample material in the nanowells (including the nanowells404A-404C) can be subjected to electromagnetic radiation from respectivelinear waveguides (including the linear waveguides 406A-406B,respectively). Emissions resulting from such exposure to electromagneticradiation (an example of emissions being fluorescence from fluorophores)can be captured using equipment (e.g., one or more cameras and/or otherimaging devices). Such equipment is sometimes referred to by way of theexpression emission equipment or a similar term. For example, emissionequipment can include one or more cameras or other image sensors and atleast one lens or other emission optics. In some implementations, thediffraction limit can be at least partially attributable to one or morecharacteristics of the emission optics. For example, based on theemission optics used, a resolution distance can be defined, theresolution distance marking the shortest distance that can be resolvedusing the emission optics. That is, when resolving features that arespaced apart by the resolution distance, the imaging system can be saidto be operating at its highest available level of resolution.

The gratings 402 serve for coupling electromagnetic radiation intoand/or out of the linear waveguides of the flowcell 400. Here, thelinear waveguide 406A has a grating 402A, the linear waveguide 406B hasa grating 402B, and a linear waveguide 406C has a grating 402C. Thegratings 402A-402C can have the same or different periodic structure. Insome implementations, either or all of the gratings 402A-402C caninclude a periodic structure of ridges interspersed by another material.For example, ridges of the gratings 402A-402C can have a pitch of about200-300 nm, to name just one example.

The gratings 402A-402C can have one or more characteristics thatfacilitate selective coupling of electromagnetic radiation into thecorresponding linear waveguide 406A-406C. In some implementations, oneor more of the gratings 402 is spatially offset from one or more othersof the gratings 402. The offset can be in a direction that is parallelto the linear waveguides 406A-406C. For example, the distance betweenthe grating 402B and the closest nanowell of the nanowells associatedwith the linear waveguide 406B is here greater than the distance betweenthe grating 402A and the closest nanowell of the nanowells associatedwith the linear waveguide 406A. As another example, the distance betweenthe grating 402C and the closest nanowell of the nanowells associatedwith the linear waveguide 406C is here greater than the distance betweenthe grating 402A and the closest nanowell of the nanowells associatedwith the linear waveguide 406A, and also greater than the distancebetween the grating 402B and the closest nanowell of the nanowellsassociated with the linear waveguide 406B. The characteristic of thegratings 402A-402C being spatially offset from each other facilitatescoupling of electromagnetic radiation (e.g., light) into one of thelinear waveguides (e.g., the linear waveguide 406A) without coupling theelectromagnetic radiation (e.g., light) into another of the linearwaveguides (e.g., the linear waveguide 406B or 406C). That is, thegrating 402C is spatially offset in the direction parallel to the linearwaveguides 406A-406C from each of the gratings 402A-402B.

In some implementations, coupling into the gratings 402A-402C can alsoor instead be differentiated by a beam parameter other than location ofthe light beam (e.g., but not limited to, angle of incidence,divergence, mode profile, polarization, aspect ratio, diameter,wavelength, and combinations thereof). In some implementations, couplinginto the gratings 402A-402C can also or instead be differentiated by acoupler parameter (e.g., but not limited to, grating period, refractiveindex, pitch, groove width, groove height, groove spacing, gratingnon-uniformity, groove orientation, groove curvature, overall shape ofthe coupler, and combinations thereof). In some implementations,coupling into the gratings 402A-402C can also or instead bedifferentiated by a waveguide parameter regarding one or more linearwaveguides of the flowcell 400 (e.g., but not limited to,cross-sectional profile, refractive index difference, mode matching withthe coupler and/or beam, and combinations thereof).

Light areas 408A-408C are here schematically illustrated as rectangleswith dashed outlines. The light areas 408A-408C represents positionswhere light or other electromagnetic radiation is caused to impinge aspart of an imaging process. In some implementations, illuminating lightgenerated by a laser can be directed at one or more of the light areas408A-408C in order to eventually be coupled into the correspondinglinear waveguide. For example, the laser light can be selected so as tocorrespond to fluorescence properties of one or more fluorophores in thesample material. Light can be directed to the light area 408A to couplelight into the linear waveguide 406A without coupling the light into thelinear waveguides 406B-406C. Light can be directed to the light area408B to couple light into the linear waveguide 406B without coupling thelight into the linear waveguides 406A or 408-408C. Light can be directedto the light area 408C to couple light into the linear waveguide 406Cwithout coupling the light into the linear waveguides 406A-406B.

The flowcell 400 can have other linear waveguides in addition to thelinear waveguides 406A-406C, with corresponding gratings. Individualgratings of such other linear waveguides can have spatial offsetssimilar to the spatial offsets of one of the gratings 402A-402C, or canhave different spatial offsets. For example, light may be coupled intoonly the first, fourth, seventh, tenth, and so on, linear waveguide,whereas light is not coupled into the second, third, fifth, sixth,eighth, ninth, eleventh, twelfth, and so on, linear waveguide. Moregenerally, in any individual scanning operation (corresponding to theuse of a particular light area, such as one of the light areas408A-408C), the ordinals of the linear waveguides into which light iscoupled can form an arithmetic series where the nth ordinal a_(n) (n=1,2, 3, . . . ) can be expressed as a_(n)=a₁+d(n−1), where a₁ is the firstordinal and d is a positive integer. For example, with a₁=1 and d=3 oneobtains that the linear waveguides into which light is coupled have theordinals 1, 4, 7, 10, and so on, corresponding to the example mentionedabove. As another example, with a₁=1 and d=4 one obtains that the linearwaveguides into which light is coupled have the ordinals 1, 5, 9, 13,and so on.

The examples described above illustrate that the flowcell 400 includes ananowell layer having first (e.g., the nanowells associated with thelinear waveguide 406A) and second (e.g., the nanowells associated withthe linear waveguide 406B) sets of nanowells to receive a sample. Theflowcell 400 includes a first linear waveguide (e.g., the linearwaveguide 406A) aligned with the first set of nanowells, and a secondlinear waveguide (e.g., the linear waveguide 406B) aligned with thesecond set of nanowells; and a first grating (e.g., the grating 402A)for the first linear waveguide, and a second grating (e.g., the grating402B-402C) for the second linear waveguide. The first grating has afirst characteristic (e.g., being spatially offset from the gratings402B-402C) to facilitate coupling of first light into the first linearwaveguide without coupling the first light into the second linearwaveguide.

FIG. 5 shows a cross section of part of an example flowcell 500. Theflowcell 500 can be used with one or more methods described herein,and/or be used in combination with one or more systems or apparatusesdescribed herein. The flowcell 500 is shown in cross section, and only aportion of the flowcell 500 is shown, for purposes of illustration.

The flowcell 500 includes a nanowell layer 502 that includes nanowells502A-502B. The nanowell layer 502 can be formed by nanoimprinting or alift-off process. For example, the nanowells 502A-502B can be formed byapplication of a nano scale template to a resin.

The flowcell 500 includes linear waveguides 504A-504B. One or more ofthe linear waveguides 504A-504B can be aligned with one or more of thenanowells 502A-502B. For example, the linear waveguide 504A is herealigned with the nanowell 502A, and the linear waveguide 504B is herealigned with the nanowell 502B.

Each of the linear waveguides 504A-504B can have one or more gratings(omitted here for clarity) to couple electromagnetic radiation intoand/or out of that linear waveguide 504A-504B. One or more directions oftravel for the electromagnetic radiation in the linear waveguides504A-504B can be employed. For example, the direction of travel can beinto and/or out of the plane of the present illustration.

Each of the linear waveguides 504A-504B can be positioned against one ormore types of cladding. The cladding can serve to constrain theelectromagnetic radiation to the respective linear waveguide 504A-504Band prevent, or reduce the extent of, propagation of the radiation intoother linear waveguides 504A-504B or other substrates (e.g., to reducecross-coupling). Here, claddings 506 are shown as an example. In someimplementations, the claddings 506 comprise a series of blocks. In someimplementations, the claddings 506 provides refractive indices thatalternate along the structure of the claddings 506. For example, a firstone of the claddings 506 can have a first refractive index, a second oneof the claddings 506 adjacent to the first one can have a secondrefractive index, a third one of the claddings 506 adjacent to thesecond one can have the first refractive index, and so on. The claddings506 can be positioned against or near the linear waveguide 102A ondifferent (e.g., opposing) sides thereof. For example, a cladding 506Acan be positioned against or near the linear waveguide 504B. Here, thecladdings 506 include multiple structures, including the cladding 506A.The claddings 506 can be made from one or more suitable materials thatserve to separate the linear waveguides 504A-504B from each other. Insome implementations, the claddings 506 can be made from a materialhaving a lower refractive index than the refractive index/indices of thelinear waveguides 504A-504B. In some implementations, one or more of thecladdings 506 includes a polymer material. In some implementations, themultiple structures of the claddings 506 can be interspersed by regionsof vacuum or another material (e.g., air or a liquid).

FIG. 6 shows an example of a flowcell 600 where multiple linearwaveguides share a common grating. The flowcell 600 can be used with oneor more methods described herein, and/or be used in combination with oneor more systems or apparatuses described herein. The flowcell 500 isshown in a top view, and only a portion of the flowcell 600 is shown,for purposes of illustration.

The flowcell 600 includes a substrate 602. In some implementations, thesubstrate 602 serves as a base layer for the flowcell 600 and cansupport one or more layers and/or other structures. For example, thesubstrate 602 can support one or more linear waveguide components 604Aand a nanowell layer (not shown).

The linear waveguide component 604A includes a coupling component 606Ahaving a grating 608A. A linear waveguide connector 610 connects thecoupling component 606A and a linear waveguide array 612A to each other.The linear waveguide array 612A includes a linear waveguide distributor614 coupled to the linear waveguide connector 610, and multiple linearwaveguides 616A arranged parallel to each other and being coupled to thelinear waveguide distributor 614. In operation, light that is incidenton the grating 608A can be coupled by the coupling component 606A andthe linear waveguide connector 610 into the linear waveguide array 612A.In the linear waveguide array 612A the linear waveguide distributor 614can distribute the light into the linear waveguides 616A. In someimplementations, the linear waveguides 616A are positioned adjacentnanowells (not shown) to facilitate imaging as part of sample analysis.For example, rows of nanowells can be positioned along each of thelinear waveguides 616A. The linear waveguide component 604A can be madefrom one or more suitable materials that facilitate propagation ofelectromagnetic radiation. In some implementations, the material(s) ofthe linear waveguide component 604A can include a polymer material. Insome implementations, the material(s) of the linear waveguide component604A can include Ta₂O₅ and/or SiN_(x).

The linear waveguide array 612A can facilitate placement of one or moreother components of the flowcell 600. In some implementations, theflowcell 600 includes a linear waveguide component 604B that ispositioned on an opposite side of the substrate 602 from the linearwaveguide array 612A. The linear waveguide component 604B can include acoupling component 606B coupled to a linear waveguide array 612B. Insome implementations, individual linear waveguides 616B of the linearwaveguide array 612B can be interspersed between the respective linearwaveguides 616A of the linear waveguide array 612A. For example, two ofthe linear waveguides 616A can be positioned on respective opposingsides of one of the linear waveguides 616B. The two of the linearwaveguides 616A are then sharing the same grating, in this example thegrating 608A of the linear waveguide component 604A.

In some implementations, the linear waveguide 616A and the linearwaveguide 616B can be positioned closer to each other than a resolutiondistance of emission optics. For example, during a first scanning stagelight can be coupled into the linear waveguides 616A of the linearwaveguide component 604A and not into the linear waveguides 616B of thelinear waveguide component 604B. During a second scanning stage,moreover, light can instead be coupled into the linear waveguides 616Bof the linear waveguide component 604B and not into the linearwaveguides 616A of the linear waveguide component 604A.

At least one of the coupling components 606A-604B can include asubstrate having an substantially, and in at least one instancecompletely, triangular shape. This can provide advantages in terms ofefficient positioning of multiple flowcell. A linear waveguide component604C may not be considered to be a part of the flowcell 600 but mayinstead be considered to be a part of another flowcell (not shown). Insome implementations, the triangular substrate of the coupling component606A, and a corresponding triangular substrate of a coupling component606C of the linear waveguide component 604C can be positioned adjacenteach other. For example, the coupling components 606A and 606C can bepositioned in opposing orientations so as to provide an efficientpacking of the linear waveguide components 604A and 604C next to eachother.

The grating 608A can be positioned toward a first end of the linearwaveguide component 604A (in this illustration, toward a left endthereof, for example). Moreover, a grating 608B can be positioned towarda second end of the linear waveguide component 604B (in thisillustration, toward a right end thereof, for example). The first endcan be positioned opposite from the second end in a direction parallelto rows of nanowells (e.g., the direction being parallel to the linearwaveguides 616A-616B).

More or fewer linear waveguide components than shown can be used. Insome implementations, respective linear waveguide components 604D-604Fare implemented. For example, the linear waveguide components 604E-604Fcan be considered part of the flowcell 600, whereas the linear waveguidecomponent 604D can be considered part of another flowcell (not shown)which is separate from the flowcell of the linear waveguide component604C.

In some implementations, coupling into the grating 608A and/or otherscan be differentiated by a beam parameter (e.g., but not limited to,location of the light beam, angle of incidence, divergence, modeprofile, polarization, aspect ratio, diameter, wavelength, andcombinations thereof). In some implementations, coupling into thegrating 608A and/or others can also or instead be differentiated by acoupler parameter (e.g., but not limited to, grating period, refractiveindex, pitch, groove width, groove height, groove spacing, gratingnon-uniformity, groove orientation, groove curvature, overall shape ofthe coupler, and combinations thereof). In some implementations,coupling into the grating 608A and/or others can also or instead bedifferentiated by a waveguide parameter regarding one or more linearwaveguides of the flowcell 600 (e.g., but not limited to,cross-sectional profile, refractive index difference, mode matching withthe coupler and/or beam, and combinations thereof).

FIG. 7 is a diagram of an example illumination system 700. Theillumination system 700 can be used with one or more methods describedherein, and/or be used in combination with one or more systems orapparatuses described herein.

The illumination system 700 includes a light source assembly 710, amirror 728, an objective lens 734, a flowcell 736, an emission dichroicfilter 738, a first optical detection subsystem 756, and a secondoptical detection subsystem 758. The illumination system 700 enablessimultaneous imaging of two color channels. In some implementations,another illumination system can be configured to enable simultaneousimaging of more than two color channels, e.g., three color channels,four color channels, or more. It is noted that there may be otheroptical configurations that can produce a similar, simultaneous imagingof multiple color channels.

The light source assembly 710 produces excitation illumination that isincident on the flowcell 736. This excitation illumination in turn willproduce emitted illumination, or fluoresced illumination, from one ormore fluorescent dyes that will be collected using the lenses 742 and748. The light source assembly 710 includes a first excitationillumination source 712 and corresponding converging lens 714, a secondexcitation illumination source 716 and corresponding converging lens718, and a dichroic filter 720.

The first excitation illumination source 712 and the second excitationillumination source 716 illustrate an illumination system that cansimultaneously provide respective excitation illumination lights for asample (e.g., corresponding to respective color channels). In someimplementations, each of the first excitation illumination source 712and the second excitation illumination source 716 includes a lightemitting diode (LED). In some implementations, at least one of the firstexcitation illumination source 712 and the second excitationillumination source 716 includes a laser. The converging lenses 714 and718 are each set a distance from the respective excitation illuminationsources 712 and 716 such that the illumination emerging from each of theconverging lenses 714/718 is focused at a field aperture 722. Thedichroic filter 720 reflects illumination from the first excitationillumination source 712 and transmits illumination from the secondexcitation illumination source 716.

In some implementations, the mixed excitation illumination output fromthe dichroic filter 720 can directly propagate toward the objective lens134. In other implementations, the mixed excitation illumination can befurther modified and/or controlled by additional intervening opticalcomponents prior to emission from the objective lens 734. The mixedexcitation illumination can pass through a focus in the field aperture722 to a filter 724 and then to a color-corrected collimating lens 726.The collimated excitation illumination from the lens 726 is incidentupon a mirror 728 upon which it reflects and is incident on anexcitation/emission dichroic filter 730. The excitation/emissiondichroic filter 730 reflects the excitation illumination emitted fromthe light source assembly 710 while permitting emission illumination,which will be described further below, to pass through theexcitation/emission dichroic filter 730 to be received by one or moreoptical subsystems 756, 758. The optical subsystems 756 and 758exemplify a light collection system that can simultaneously collectmultiplexed fluorescent light. The excitation illumination reflectedfrom the excitation/emission dichroic filter 730 is then incident upon amirror 732, from which it is incident upon the objective lens 134towards the flowcell 736.

The objective lens 734 focuses the collimated excitation illuminationfrom the mirror 732 onto the flowcell 136. In some implementations, theobjective lens 734 is a microscope objective with a specifiedmagnification factor of, for example, 1×, 2×, 4×, 5×, 6×, 8×, 10×, orhigher. The objective lens 734 focuses the excitation illuminationincident from the mirror 732 onto the flowcell 736 in a cone of angles,or numerical aperture, determined by the magnification factor. In someimplementations, the objective lens 734 is movable on an axis that isnormal to the flow cell (a “z-axis”). In some implementations, theillumination system 700 adjusts the z position of the tube lens 748 andtube lens 742 independently.

The flowcell 736 contains a sample, such as a nucleotide sequence or anyother material, to be analyzed. The flowcell 736 can include one or morechannels 760 (here schematically illustrated by way of a cross-sectionview in an enlargement) configured to hold sample material and tofacilitate actions to be taken with regard to the sample material,including, but not limited to, triggering chemical reactions or addingor removing material. An object plane 762 of the objective lens 734,here schematically illustrated using a dashed line, extends through theflowcell 736. For example, the object plane 762 can be defined so as tobe adjacent to the channel(s) 760.

The objective lens 734 can define a field of view. The field of view candefine the area on the flowcell 736 from which an image detectorcaptures emitted light using the objective lens 734. One or more imagedetectors, e.g., detectors 746 and 754, can be used. The illuminationsystem 700 can include separate image detectors 746 and 754 for therespective wavelengths (or wavelength ranges) of the emitted light. Atleast one of the image detectors 746 and 754 can include acharge-coupled device (CCD), such as a time-delay integration CCDcamera, or a sensor fabricated based on complementary metal-oxidesemiconductor (CMOS) technology, such as chemically sensitive fieldeffect transistors (chemFET), ion-sensitive field effect transistors(ISFET), and/or metal oxide semiconductor field effect transistors(MOSFET).

In some implementations, the illumination system 700 can include astructured illumination microscope (SIM). SIM imaging is based onspatially structured illumination light and reconstruction to result ina higher resolution image than an image produced solely using themagnification from the objective lens 734. For example, the structurecan consist of or include a pattern or grating that interrupts theilluminating excitation light. In some implementations, the structurecan include patterns of fringes. Fringes of light can be generated byimpinging a light beam on a diffraction grating such that reflective ortransmissive diffraction occurs. The structured light can be projectedonto the sample, illuminating the sample according to the respectivefringes which may occur according to some periodicity. To reconstruct animage using SIM, the two or more patterned images are used where thepattern of excitation illumination are at different phase angles to eachother. For example, images of the sample can be acquired at differentphases of the fringes in the structured light, sometimes referred to asthe respective pattern phases of the images. This can allow variouslocations on the sample to be exposed to a multitude of illuminationintensities. The set of resulting emitted light images can be combinedto reconstruct the higher resolution image.

The sample material in the flowcell 736 is contacted with fluorescentdyes that couple to corresponding nucleotides. The fluorescent dyes emitfluorescent illumination upon being irradiated with correspondingexcitation illumination incident on the flowcell 736 from the objectivelens 734. The emitted illumination is identified with wavelength bands,each of which can be categorized to a respective color channel. Thefluorescent dyes are chemically conjoined with respective nucleotides,e.g., containing respective nucleobases. In this way, a dNTP labeledwith a fluorescent dye may be identified based upon an emitted lightwavelength being within a corresponding wavelength band when detected byan image detector 746, 754.

The objective lens 734 captures fluorescent light emitted by thefluoresced dye molecules in the flow cell 736. Upon capturing thisemitted light, the objective lens 734 collects and conveys collimatedlight. This emitted light then propagates back along the path in whichthe original, excitation illumination arrived from the light sourceassembly 710. It is noted that there is little to no interferenceexpected between the emitted and excitation illumination along this pathbecause of the lack of coherence between the emitted light andexcitation illumination. That is, the emitted light is a result of aseparate source, namely that of the fluorescent dye in contact with thesample material in the flowcell 736.

The emitted light, upon reflection by the mirror 732, is incident on theexcitation/emission dichroic filter 730. The filter 730 transmits theemitted light to a dichroic filter 738.

In some implementations, a dichroic filter 738 transmits illuminationassociated with the blue color channel and reflects illuminationassociated with the green color channel. In some implementations, thedichroic filter 738 is selected such that the dichroic filter 738reflects emitted illumination to an optical subsystem 756 that is withinthe defined green wavelength band and transmits emitted illumination toan optical subsystem 758 that is within the defined blue wavelengthband, as discussed above. The optical subsystem 756 includes a tube lens742, a filter 744, and the image detector 746. The optical subsystem 758includes a tube lens 748, a filter 750, and the image detector 754.

In some implementations, the dichroic filter 738 and the dichroic filter7120 operate similarly to each other (e.g., both may reflect light ofone color and transmit light of another color). In otherimplementations, the dichroic filter 738 and the dichroic filter 720operate differently from each other (e.g., the dichroic filter 738 maytransmit light of a color that the dichroic filter 720 reflects, andvice versa).

In some implementations, the emitted illumination encounters a mirror752 prior to the image detector 754. In example shown, the optical pathin the optical subsystem 758 is angled so that the illumination system700 as a whole may satisfy space or volume requirements. In someimplementations, both such subsystems 756 and 758 have optical pathsthat are angled. In some implementations, neither of the optical pathsin the subsystem 756 nor 758 is angled. As such, one or more of multipleoptical subsystems can have at least one angled optical path.

Each tube lens 742 and 748 focuses the emitted illumination incidentupon it onto respective image detectors 746 and 754. Each detector 746and 754 includes, in some implementations, a CCD array. In someimplementations, each image detector 746 and 754 includes acomplementary metal-oxide semiconductor (CMOS) sensor.

The illumination system 700 is not required to be as shown in FIG. 7.For example, each of the mirrors 728, 732, 740 may be replaced with aprism or some other optical device that changes the direction ofillumination. Each lens may be replaced with a diffraction grating, adiffractive optic, a Fresnel lens, or some other optical device thatproduces collimated or focused illumination from incident illumination.

FIGS. 8-9 are flowcharts of example methods 800 and 900. The method 800or 900, or both, can be performed using, and/or in combination with, oneor more other examples described herein. More or fewer operations can beperformed, and/or two or more operations can be performed in a differentorder, unless otherwise indicated.

At 810, a sample can be applied to first and second rows of nanowells ofa flowcell. In some implementations, the sample can be applied to therows of nanowells associated with the linear waveguides 206A-206B inFIG. 2A. In some implementations, the sample can be applied to the rowsof nanowells associated with the linear waveguides 406A-406B in FIG. 4.For example, the sample can include genetic material.

At 820, a position of an illumination component can be changed toaddress a subset of gratings. In some implementations, the position ofthe illumination component is changed so that illuminating light will ordoes impinge on the light area 214 in FIG. 2A, when the light area 214is aligned with the gratings 202A and 202C and some others, but not withthe grating 202B and some others. In some implementations, the positionof the illumination component is changed so that illuminating light willor does impinge on the light area 408A in FIG. 4, which is aligned withthe grating 402A, but not with the gratings 402B-402C. For example, themirror 732 in FIG. 7 can be adjusted to change the location where lightis incident. In some implementations, the flowcell can be moved oradjusted in addition to, or in lieu of, moving the illuminationequipment.

At 830, scanning can begin in a first direction. In someimplementations, scanning is performed in the direction corresponding tothe arrow 218 in FIG. 2A. The positioning can include movement of animage capture area (e.g., movement of image capture apparatus), or theflowcell, or both.

At 840, first light can be directed at a first grating of a first linearwaveguide aligned with the first row of nanowells, without coupling thefirst light into a second linear waveguide aligned with the second rowof nanowells. In some implementations, first light is directed at thelight area 214 in FIG. 2A, when the light area 214 at least in partoverlays the gratings 202A and 202C. Because the grating 202B isspatially offset from the gratings 202A and 202C, the first light is notcoupled into the linear waveguide 206B. In some implementations, firstlight is directed at the light area 408A in FIG. 4 which at least inpart overlays the grating 402A. Because the gratings 402B-402C arespatially offset from the grating 402A, the first light is not coupledinto the linear waveguides 406B-406C.

At 850, one or more images can be captured. In some implementations, animage can be captured of the image capture area 216 in FIG. 2A when theimage capture area 216 at least in part overlays some aspect of theflowcell 200. In a similar way, one or more images can be captured ofthe flowcell 400 in FIG. 4. For example, image capture can include aline scan.

At 860, a position of the illumination component can be changed toaddress another subset of gratings. In some implementations, theposition of the illumination component is changed so that illuminatinglight will or does impinge on the light area 214 in FIG. 2B, when thelight area 214 is aligned with the grating 202B and some others, but notwith the gratings 202A and 202C and some others. In someimplementations, the position of the illumination component is changedso that illuminating light will or does impinge on the light area 408Bin FIG. 4, which is aligned with the grating 402B, but not with thegratings 402A or 402C. For example, the mirror 732 in FIG. 7 can beadjusted to change the location where light is incident. In someimplementations, the flowcell can be moved or adjusted in addition to,or in lieu of, moving the illumination equipment.

At 870, scanning can begin in a second direction. The second directioncan be the same as, or different from, the first direction. In someimplementations, scanning is performed in the direction corresponding tothe arrow 220 in FIG. 2B. The positioning can include movement of animage capture area (e.g., movement of image capture apparatus), or theflowcell, or both.

At 880, second light can be directed at a second grating of a secondlinear waveguide aligned with the second row of nanowells, withoutcoupling the second light into the first linear waveguide. In someimplementations, the second light is directed at the light area 214 inFIG. 2B, when the light area 214 at least in part overlays the grating202B. Because the gratings 202A and 202C are spatially offset from thegrating 202B, the second light is not coupled into the linear waveguides206A or 206C. In some implementations, the second light is directed atthe light area 408B in FIG. 4, when the light area 408B at least in partoverlays the grating 402B. Because the gratings 402A and 402C arespatially offset from the grating 402B, the second light is not coupledinto the linear waveguides 406A and 406C.

At 890, one or more images can be captured. In some implementations, animage can be captured of the image capture area 216 in FIG. 2B when theimage capture area 216 at least in part overlays some aspect of theflowcell 200. In a similar way, one or more images can be captured ofthe flowcell 400 in FIG. 4. For example, image capture can include aline scan.

Turning now to the method 900 in FIG. 9, at 910 a sample can be appliedto first and second rows of nanowells of a flowcell. In someimplementations, the sample can be applied to the rows of nanowellsassociated with the linear waveguides 306A-306B in FIG. 3A. For example,the sample can include genetic material.

At 920, a position of an illumination component can be changed to anangle associated with a grating period of a subset of gratings. In someimplementations, the position of the illumination component is changedso that illuminating light will or does have an incident angle at whichthe gratings 302A and 302C and some others couple light, but at whichthe grating 302B and some others do not couple light. For example, themirror 732 in FIG. 7 can be adjusted to change the incident angle. Insome implementations, the flowcell can be moved or adjusted in additionto, or in lieu of, adjusting the illumination equipment.

At 930, scanning can begin in a first direction. In someimplementations, scanning is performed in the direction corresponding tothe arrow 318 in FIG. 3A. The positioning can include movement of animage capture area (e.g., movement of image capture apparatus), or theflowcell, or both.

At 940, first light can be directed at a first grating of a first linearwaveguide aligned with the first row of nanowells, without coupling thefirst light into a second linear waveguide aligned with the second rowof nanowells. In some implementations, the first light is directed atthe light area 314 in FIG. 3A when the light area 314 at least partiallyoverlaps the gratings 302. Because the grating 302B has a differentgrating period than the gratings 302A and 302C, the first light is notcoupled into the linear waveguide 306B.

At 950, one or more images can be captured. In some implementations, animage can be captured of the image capture area 316 in FIG. 3A when theimage capture area 316 at least in part overlays some aspect of theflowcell 300.

At 960, a position of the illumination component can be changed to anangle associated with a grating period of another subset of gratings. Insome implementations, the position of the illumination component ischanged so that illuminating light will or does have an incident angleat which the grating 302B and some others couple light, but at which thegratings 302A and 302C and some others do not couple light. For example,the mirror 732 in FIG. 7 can be adjusted to change the location wherelight is incident. In some implementations, the flowcell can be moved oradjusted in addition to, or in lieu of, moving the illuminationequipment.

At 970, scanning can begin in a second direction. The second directioncan be the same as, or different from, the first direction. In someimplementations, scanning is performed in the direction corresponding tothe arrow 320 in FIG. 3B. The positioning can include movement of animage capture area (e.g., movement of image capture apparatus), or theflowcell, or both.

At 980, second light can be directed at a second grating of a secondlinear waveguide aligned with the second row of nanowells, withoutcoupling the second light into the first linear waveguide. In someimplementations, the second light is directed at the light area 314 inFIG. 3B, when the light area 314 at least partially overlaps thegratings 302. Because the gratings 302A and 302C have different gratingperiods than the grating 302B, the second light is not coupled into thelinear waveguides 306A or 306C.

At 990, one or more images can be captured. In some implementations, animage can be captured of the image capture area 316 in FIG. 3B when theimage capture area 316 at least in part overlays some aspect of theflowcell 300. For example, image capture can include a line scan.

Some examples herein show nanowells having circular openings, forpurposes of illustration only. In some implementations, non-circularnanowells can be used. FIG. 10A shows an example of a hexagonal array1000 of non-circular nanowells 1002. The hexagonal array 1000 can beused with one or more methods described herein, and/or be used incombination with one or more systems or apparatuses described herein.For example, the hexagonal array 1000 can be used with circularnanowells or non-circular nanowells, or both. One or more of thenon-circular nanowells 1002 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. For example, the nanowells 1002 can bearranged in a hexagonal array or a non-hexagonal (e.g., otherwisepolygonal) array, or both.

The size and/or shape of the non-circular nanowells 1002 can affect theimaging that is part of the analysis process. In some implementations, afluorescence signal is collected from some or all of the non-circularnanowells 1002. The fluorescence signal can be affected by the sizeand/or shape of the non-circular nanowells 1002. For example, changes inthe fluorescence signal(s) generated can affect the throughput of ananalysis system (e.g., a sequencing system).

In some implementations, one or more of the non-circular nanowells 1002has an elliptical opening. An ellipsis can be characterized by therespective lengths of the major and minor axes. The length of the minoraxis can be expressed as a percentage of the major axis length,including, but not limited to, as having 5%, 15%, 35%, 65% or 95%, ofthe length of the major axis, to name just a few examples. Othergeometries than elliptical of the non-circular nanowells are alsopossible.

FIG. 10B shows an example of a triangular array 1004 of circularnanowells 1006. The triangular array 1004 can be used with one or moremethods described herein, and/or be used in combination with one or moresystems or apparatuses described herein. For example, the triangulararray 1004 can be used with circular nanowells or non-circularnanowells, or both. One or more of the circular nanowells 1006 can beused with one or more methods described herein, and/or be used incombination with one or more systems or apparatuses described herein.For example, the circular nanowells 1006 can be arranged in a hexagonalarray or a non-hexagonal (e.g., otherwise polygonal) array, or both.

FIG. 11 shows another example of a flowcell 1100 having staggeredgratings 1102. The flowcell 1100 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. For example, the flowcell 1100 can beused with staggered gratings or non-staggered gratings, or both. One ormore of the staggered gratings 1102 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. For example, the staggered gratings1102 can be used with nanowells arranged in a hexagonal array or anon-hexagonal (e.g., otherwise polygonal) array, or both.

The flowcell 1100 includes nanowells, including a nanowell 1104A, thatare here illustrated using circular shapes. Only some of the nanowellswill be specifically mentioned, and the other nanowells may be similaror identical to the one(s) discussed. The nanowells may be formed in ananowell layer (e.g., by way of nanoimprinting or a lift-off process).For example, the nanowells can be formed in a resin using a nanoscaletemplate. The nanowell layer is not explicitly shown in this example,for purposes of clarity. The nanowell 1104A is here associated with alinear waveguide 1106A. In some implementations, the linear waveguidesdescribed with reference to the flowcell 1100 can be similar oridentical to one or more other linear waveguides described herein. Forexample, the linear waveguide 1106A is positioned adjacent (e.g., incontact with or near) to the nanowell layer that includes the nanowell1104A.

Another nanowell 1104B is also associated with the linear waveguide1106A. For example, the nanowell 1104B is positioned adjacent to thenanowell 1104A and both of the nanowells 1104A-1104B can interact withthe linear waveguide 1106A in an imaging process (e.g., by way ofreceiving electromagnetic radiation from the linear waveguide 1106A).Another nanowell 1104C, by contrast, is instead associated with a linearwaveguide 1106B. In some implementations, the linear waveguide 1106B ispositioned adjacent to the linear waveguide 1106A. For example, cladding(not shown) and/or another material can be positioned between the linearwaveguides 1106A-1106B.

A nanowell 1104D is here associated with a linear waveguide 1106C. Insome implementations, the linear waveguide 1106C is positioned adjacentto the linear waveguide 1106B. For example, cladding (not shown) and/oranother material can be positioned between the linear waveguides1106B-1106C.

A nanowell 1104E is here associated with a linear waveguide 1106D. Insome implementations, the linear waveguide 1106D is positioned adjacentto the linear waveguide 1106C. For example, cladding (not shown) and/oranother material can be positioned between the linear waveguides1106C-1106D.

The nanowells 1104A-1104B and others here form a first set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1106A. The nanowell 1104C and others here form a second set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1106B. The nanowell 1104D and others here form a third set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1106C. The nanowell 1104E and others here form a fourth set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1106D. In some implementations, the first set of nanowells (e.g., thenanowells 1104A-1104B and others) is positioned so as to be in phasewith the second set of nanowells (e.g., the nanowells 1104C and others).The first set of nanowells can be positioned at substantially, and in atleast one instance completely, regular intervals along the linearwaveguide 1106A. For example, each of the nanowells in the first set ofnanowells at the linear waveguide 1106A has a corresponding nanowell inthe second set of nanowells at the linear waveguide 1106B. Thecorresponding nanowell may be positioned directly across the cladding oranother material between the linear waveguides 1106A-1106B from thenanowell.

Here, the nanowell 1104D and the others in the third set of nanowellsare positioned at substantially, and in at least one instancecompletely, regular intervals along the linear waveguide 1106C. Thethird set of nanowells is positioned so as to be out of phase with atleast the second set of nanowells. In some implementations, none of thenanowells in the second set of nanowells has a corresponding nanowell inthe third set of nanowells directly across the cladding or othermaterial. For example, each of the nanowells in the second set ofnanowells may be equidistantly positioned between two adjacent ones ofthe nanowells in the third set of nanowells.

In some implementations, the fourth set of nanowells (e.g., the nanowell1104E and others along the linear waveguide 1106D) is positioned so asto be in phase with the third set of nanowells (e.g., the nanowells1104D and others along the linear waveguide 1106C). The fourth set ofnanowells can be positioned at substantially, and in at least oneinstance completely, regular intervals along the linear waveguide 1106D.For example, each of the nanowells in the fourth set of nanowells at thelinear waveguide 1106D has a corresponding nanowell in the third set ofnanowells at the linear waveguide 1106C. The corresponding nanowell maybe positioned directly across the cladding or another material betweenthe linear waveguides 1106C-1106D from the nanowell.

The gratings 1102 serve for coupling electromagnetic radiation intoand/or out of the linear waveguides of the flowcell 1100. Here, thelinear waveguide 1106A has a grating 1102A, the linear waveguide 1106Bhas a grating 1102B, the linear waveguide 1106C has a grating 1102C, andthe linear waveguide 1106D has a grating 1102D. Each of the gratings1102A-1102D can have the same or different periodic structure. In someimplementations, some or all of the gratings 1102A-1102D can include aperiodic structure of ridges interspersed by another material. Forexample, ridges of the gratings 1102A-1102D can have a pitch of about200-300 nm, to name just one example.

The gratings 1102A-1102D can have one or more characteristics that atleast in part facilitate selective coupling of electromagnetic radiationinto the corresponding linear waveguide 1106A-1106D. In someimplementations, one or more of the gratings 1102 is spatially offsetfrom one or more others of the gratings 1102. The offset can be in adirection that is parallel to the linear waveguides 1106A-1106D. Forexample, the distance between the grating 1102B and the closest nanowellof the nanowells associated with the linear waveguide 1106B is heregreater than the distance between the grating 1102A and the closestnanowell of the nanowells associated with the linear waveguide 1106A. Asanother example, the distance between the grating 1102D and the closestnanowell of the nanowells associated with the linear waveguide 1106D ishere greater than the distance between the grating 1102C and the closestnanowell of the nanowells associated with the linear waveguide 1106C. Insome implementations, the gratings 1102A and 1102C have the same or asimilar spatial offset. In some implementations, the gratings 1102B and1102D have the same or a similar spatial offset. The characteristic ofthe gratings 1102A-1102D being spatially offset from each other at leastin part facilitates coupling of electromagnetic radiation (e.g., light)into one of the linear waveguides (e.g., the linear waveguide 1106Aand/or 1106C) without coupling the electromagnetic radiation (e.g.,light) into another of the linear waveguides (e.g., the linear waveguide1106B and/or 1106D).

In some implementations, coupling into the gratings 1102A-1102D can alsoor instead be differentiated by a beam parameter other than location ofthe light beam (e.g., but not limited to, angle of incidence,divergence, mode profile, polarization, aspect ratio, diameter,wavelength, and combinations thereof). In some implementations, couplinginto the gratings 1102A-1102D can also or instead be differentiated by acoupler parameter (e.g., but not limited to, grating period, refractiveindex, pitch, groove width, groove height, groove spacing, gratingnon-uniformity, groove orientation, groove curvature, overall shape ofthe coupler, and combinations thereof). In some implementations,coupling into the gratings 1102A-1102D can also or instead bedifferentiated by a waveguide parameter regarding one or more of thelinear waveguides 1106A-1106D (e.g., but not limited to, cross-sectionalprofile, refractive index difference, mode matching with the couplerand/or beam, and combinations thereof).

The examples described above illustrate that the flowcell 1100 includesa nanowell layer having first (e.g., the nanowells associated with thelinear waveguide 1106A) and second (e.g., the nanowells associated withthe linear waveguide 1106B) sets of nanowells to receive a sample. Theflowcell 1100 includes a first linear waveguide (e.g., the linearwaveguide 1106A) aligned with the first set of nanowells, and a secondlinear waveguide (e.g., the linear waveguide 1106B) aligned with thesecond set of nanowells; and a first grating (e.g., the grating 1102A)for the first linear waveguide, and a second grating (e.g., the grating1102B) for the second linear waveguide. The first grating has a firstcharacteristic (e.g., being spatially offset from the grating 1102B) tofacilitate coupling of first light into the first linear waveguidewithout coupling the first light into the second linear waveguide.

FIG. 12 shows another example of a flowcell 1200 having staggeredgratings 1202. The flowcell 1200 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. For example, the flowcell 1200 can beused with staggered gratings or non-staggered gratings, or both. One ormore of the staggered gratings 1202 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. For example, the staggered gratings1202 can be used with nanowells arranged in a hexagonal array or anon-hexagonal (e.g., otherwise polygonal) array, or both.

The flowcell 1200 includes nanowells, including a nanowell 1204A, thatare here illustrated using circular shapes. Only some of the nanowellswill be specifically mentioned, and the other nanowells may be similaror identical to the one(s) discussed. The nanowells may be formed in ananowell layer (e.g., by way of nanoimprinting or a lift-off process).For example, the nanowells can be formed in a resin using a nanoscaletemplate. The nanowell layer is not explicitly shown in this example,for purposes of clarity. The nanowell 1204A is here associated with alinear waveguide 1206A. In some implementations, the linear waveguidesdescribed with reference to the flowcell 1200 can be similar oridentical to one or more other linear waveguides described herein. Forexample, the linear waveguide 1206A is positioned adjacent (e.g., incontact with or near) the nanowell layer that includes the nanowell1204A.

Another nanowell 1204B is also associated with the linear waveguide1206A. For example, the nanowell 1204B is positioned adjacent to thenanowell 1204A and both of the nanowells 1204A-1204B can interact withthe linear waveguide 1206A in an imaging process (e.g., by way ofreceiving electromagnetic radiation from the linear waveguide 1206A).Another nanowell 1204C, by contrast, is instead associated with a linearwaveguide 1206B. In some implementations, the linear waveguide 1206B ispositioned adjacent to the linear waveguide 1206A. For example, cladding(not shown) and/or another material can be positioned between the linearwaveguides 1206A-1206B.

A nanowell 1204D is here associated with a linear waveguide 1206C. Insome implementations, the linear waveguide 1206C is positioned adjacentto the linear waveguide 1206B. For example, cladding (not shown) and/oranother material can be positioned between the linear waveguides1206B-1206C.

A nanowell 1204E is here associated with a linear waveguide 1206D. Insome implementations, the linear waveguide 1206D is positioned adjacentto the linear waveguide 1206C. For example, cladding (not shown) and/oranother material can be positioned between the linear waveguides1206C-1206D.

The nanowells 1204A-1204B and others here form a first set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1206A. The nanowell 1204C and others here form a second set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1206B. The nanowell 1204D and others here form a third set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1206C. The nanowell 1204E and others here form a fourth set of nanowells(e.g., a row of nanowells) that extends along the linear waveguide1206D. In some implementations, the first set of nanowells (e.g., thenanowells 1204A-1204B and others) is positioned so as to be out of phasewith the second set of nanowells (e.g., the nanowells 1204C and others).In some implementations, none of the nanowells in the first set ofnanowells has a corresponding nanowell in the second set of nanowellsdirectly across the cladding or other material. For example, each of thenanowells in the first set of nanowells may be equidistantly positionedbetween two adjacent ones of the nanowells in the second set ofnanowells.

Here, the nanowell 1204D and the others in the third set of nanowellsare positioned at substantially, and in at least one instancecompletely, regular intervals along the linear waveguide 1206C. Thethird set of nanowells is positioned so as to be out of phase with atleast the second set of nanowells. In some implementations, none of thenanowells in the third set of nanowells has a corresponding nanowell inthe second set of nanowells directly across the cladding or othermaterial. For example, each of the nanowells in the third set ofnanowells may be equidistantly positioned between two adjacent ones ofthe nanowells in the second set of nanowells. The third set of nanowellscan be positioned so as to be in phase with at least the first set ofnanowells.

In some implementations, the fourth set of nanowells (e.g., the nanowell1204E and others along the linear waveguide 1206D) is positioned so asto be out of phase with the third set of nanowells (e.g., the nanowells1204D and others along the linear waveguide 1206C). In someimplementations, none of the nanowells in the fourth set of nanowellshas a corresponding nanowell in the third set of nanowells directlyacross the cladding or other material. For example, each of thenanowells in the fourth set of nanowells may be equidistantly positionedbetween two adjacent ones of the nanowells in the third set ofnanowells.

The gratings 1202 serve for coupling electromagnetic radiation intoand/or out of the linear waveguides of the flowcell 1200. Here, thelinear waveguide 1206A has a grating 1202A, the linear waveguide 1206Bhas a grating 1202B, the linear waveguide 1206C has a grating 1202C, andthe linear waveguide 1206D has a grating 1202D. Each of the gratings1202A1202D can have the same or different periodic structure. In someimplementations, some or all of the gratings 1202A-1202D can include aperiodic structure of ridges interspersed by another material. Forexample, ridges of the gratings 1202A-1202D can have a pitch of about200-300 nm, to name just one example. The gratings 1202A-1202D can haveone or more of multiple suitable shapes. In some implementations, thegratings 1202A-1202D have a truncated triangular shape.

The gratings 1202A-1202D can have one or more characteristics that atleast in part facilitate selective coupling of electromagnetic radiationinto the corresponding linear waveguide 1206A-1206D. In someimplementations, one or more of the gratings 1202 is spatially offsetfrom one or more others of the gratings 1202. The offset can be in adirection that is parallel to the linear waveguides 1206A-1206D. Forexample, the distance between the grating 1202B and the other end of thelinear waveguide 1206B is here shorter than the distance between thegrating 1202A and the other end of the linear waveguide 1206A. Asanother example, the distance between the grating 1202D and the otherend of the linear waveguide 1206D is here shorter than the distancebetween the grating 1202C and the other end of the linear waveguide1206C. In some implementations, the gratings 1202A and 1202C have thesame or a similar spatial offset. In some implementations, the gratings1202B and 1202D have the same or a similar spatial offset. Thecharacteristic of the gratings 1202A-1202D being spatially offset fromeach other at least in part facilitates coupling of electromagneticradiation (e.g., light) into one of the linear waveguides (e.g., thelinear waveguide 1206A and/or 1206C) without coupling theelectromagnetic radiation (e.g., light) into another of the linearwaveguides (e.g., the linear waveguide 1206B and/or 1206D).

In some implementations, coupling into the gratings 1202A-1202D can alsoor instead be differentiated by a beam parameter other than location ofthe light beam (e.g., but not limited to, angle of incidence,divergence, mode profile, polarization, aspect ratio, diameter,wavelength, and combinations thereof). In some implementations, couplinginto the gratings 1202A-1202D can also or instead be differentiated by acoupler parameter (e.g., but not limited to, grating period, refractiveindex, pitch, groove width, groove height, groove spacing, gratingnon-uniformity, groove orientation, groove curvature, overall shape ofthe coupler, and combinations thereof). In some implementations,coupling into the gratings 1202A-1202D can also or instead bedifferentiated by a waveguide parameter regarding one or more of thelinear waveguides 1206A-1206D (e.g., but not limited to, cross-sectionalprofile, refractive index difference, mode matching with the couplerand/or beam, and combinations thereof).

The flowcell 1200 can have the nanowells arranged in any of multiplepatterns. In the present example, the nanowells are arranged in ahexagonal array. A hexagonal array forms one or more hexagons. Here, thelinear waveguide 1206B further includes nanowells 1204F-1204G, and thelinear waveguide 1206C further includes a nanowell 1204H. The nanowells1204A1204H are here positioned in form of a hexagon. The nanowells1204A-1204B are here part of the first set of nanowells and areassociated with the linear waveguide 1206A; the nanowells 1204C and1204F-1204G are part of the second set of nanowells and are associatedwith the linear waveguide 1206B; the nanowells 1204D and 1204H are partof the third set of nanowells and are associated with the linearwaveguide 1206C.

The examples described above illustrate that the flowcell 1200 includesa nanowell layer having first (e.g., the nanowells associated with thelinear waveguide 1206A) and second (e.g., the nanowells associated withthe linear waveguide 1206B) sets of nanowells to receive a sample. Theflowcell 1200 includes a first linear waveguide (e.g., the linearwaveguide 1206A) aligned with the first set of nanowells, and a secondlinear waveguide (e.g., the linear waveguide 1206B) aligned with thesecond set of nanowells; and a first grating (e.g., the grating 1202A)for the first linear waveguide, and a second grating (e.g., the grating1202B) for the second linear waveguide. The first grating has a firstcharacteristic (e.g., being spatially offset from the grating 1202B) tofacilitate coupling of first light into the first linear waveguidewithout coupling the first light into the second linear waveguide.

FIG. 13 shows another example of a flowcell 1300 having staggeredgratings 1302. The flowcell 1300 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. For example, the flowcell 1300 can beused with staggered gratings or non-staggered gratings, or both. One ormore of the staggered gratings 1302 can be used with one or more methodsdescribed herein, and/or be used in combination with one or more systemsor apparatuses described herein. For example, the staggered gratings1302 can be used with nanowells arranged in a hexagonal array or anon-hexagonal (e.g., otherwise polygonal) array, or both.

The flowcell 1300 includes nanowells, including a nanowell 1304A, thatare here illustrated using circular shapes. Only some of the nanowellswill be specifically mentioned, and the other nanowells may be similaror identical to the one(s) discussed. The nanowells may be formed in ananowell layer (e.g., by way of nanoimprinting or a lift-off process).For example, the nanowells can be formed in a resin using a nanoscaletemplate. The nanowell layer is not explicitly shown in this example,for purposes of clarity. The nanowell 1304A is here associated with alinear waveguide 1306A. In some implementations, the linear waveguidesdescribed with reference to the flowcell 1300 can be similar oridentical to one or more other linear waveguides described herein. Forexample, the linear waveguide 1306A is positioned adjacent (e.g., incontact with or near) to the nanowell layer that includes the nanowell1304A. The nanowell 1304A is part of a first set of nanowells (e.g., oneor more rows of nanowells) for the linear waveguide 1306A. Here, the rowof nanowells of which the nanowell 1304A is part extends along thelinear waveguide 1306A on one side thereof. For example, the row ofnanowells does not overlap the linear waveguide 1306A in the shownperspective of the flowcell 1300.

Another nanowell 1304B is also associated with the linear waveguide1306A. Like the nanowell 1304A, the nanowell 1304B is also part of thefirst set of nanowells (e.g., one or more rows of nanowells) for thelinear waveguide 1306A. Here, the row of nanowells of which the nanowell1304B is part extends along the linear waveguide 1306A on another sidethereof. For example, the row of nanowells does not overlap the linearwaveguide 1306A in the shown perspective of the flowcell 1300 and ispositioned on an opposite side of the linear waveguide 1306A from therow of the nanowell 1304A. Both of the nanowells 1304A-1304B caninteract with the linear waveguide 1306A in an imaging process (e.g., byway of receiving electromagnetic radiation from the linear waveguide1306A).

Another nanowell 1304C is associated with a linear waveguide 1306B. Insome implementations, the linear waveguide 1306B is parallel to andpositioned adjacent to the linear waveguide 1306A. For example, cladding(not shown) and/or another material can be positioned between the linearwaveguides 1306A-1306B. The nanowell 1304C is part of a second set ofnanowells (e.g., one or more rows of nanowells) for the linear waveguide1306B. Here, the row of nanowells of which the nanowell 1304C is partextends along the linear waveguide 1306B on one side thereof. Forexample, the row of nanowells does not overlap the linear waveguide1306B in the shown perspective of the flowcell 1300. Another row ofnanowells that is also part of the second set of nanowells can bepositioned on the opposite side of the linear waveguide 1306B from therow of the nanowell 1304C.

Another nanowell 1304D is associated with a linear waveguide 1306C. Insome implementations, the linear waveguide 1306C is parallel to andpositioned adjacent to the linear waveguide 1306B. For example, cladding(not shown) and/or another material can be positioned between the linearwaveguides 1306B-1306C. The nanowell 1304D is part of a third set ofnanowells (e.g., one or more rows of nanowells) for the linear waveguide1306C. Here, the row of nanowells of which the nanowell 1304D is partextends along the linear waveguide 1306C on one side thereof. Forexample, the row of nanowells does not overlap the linear waveguide1306C in the shown perspective of the flowcell 1300. Another row ofnanowells that is also part of the third set of nanowells can bepositioned on the opposite side of the linear waveguide 1306C from therow of the nanowell 1304D.

Having nanowells positioned with offsets from the associated linearwaveguide (e.g., as in the flowcell 1300) can provide one or moreadvantages. In some implementations, cross-talk between waveguides canbe reduced or minimized. For example, this benefit can outweigh asomewhat lower packing density of the nanowells.

In some implementations, the nanowells in the rows of the first set ofnanowells (e.g., the nanowells 1304A-1304B and others) are positioned soas to be in phase with each other. The nanowells in the nanowell rows oneither side of the linear waveguide 1306A can be positioned atsubstantially, and in at least one instance completely, regularintervals along the linear waveguide 1306A. For example, each of thenanowells in one of these rows has a corresponding nanowell in the otherrow. The corresponding nanowell of the first set of nanowells may bepositioned directly across the linear waveguides 1306A from the othernanowell of the first set of nanowells.

In some implementations, the first set of nanowells (e.g., the nanowells1304A-1304B and others) are positioned so as to be in phase withnanowells of the second set of nanowells (e.g., the nanowell 1304C andothers). The nanowells in the nanowell rows on either side of the linearwaveguide 1306B can be positioned at substantially, and in at least oneinstance completely, regular intervals along the linear waveguide 1306B.For example, each of the nanowells in at least one of these rows has acorresponding nanowell in at least one of the rows of the first set ofnanowells. The corresponding nanowell of the first set of nanowells maybe positioned directly across the cladding or other material from thenanowell of the second set of nanowells.

The gratings 1302 serve for coupling electromagnetic radiation intoand/or out of the linear waveguides of the flowcell 1300. Here, thelinear waveguide 1306A has a grating 1302A, the linear waveguide 1306Bhas a grating 1302B, the linear waveguide 1306C has a grating 1302C, anda linear waveguide 1306D has a grating 1302D. Each of the gratings1302A-1302D can have the same or different periodic structure. In someimplementations, some or all of the gratings 1302A-1302D can include aperiodic structure of ridges interspersed by another material. Forexample, ridges of the gratings 1302A-1302D can have a pitch of about200-300 nm, to name just one example.

The gratings 1302A-1302D can have one or more characteristics that atleast in part facilitate selective coupling of electromagnetic radiationinto the corresponding linear waveguide 1306A-1306D. In someimplementations, one or more of the gratings 1302 is spatially offsetfrom one or more others of the gratings 1302. The offset can be in adirection that is parallel to the linear waveguides 1306A-1306D. Forexample, the distance between the grating 1302B and the closest nanowellof the nanowells associated with the linear waveguide 1306B is heregreater than the distance between the grating 1302A and the closestnanowell of the nanowells associated with the linear waveguide 1306A. Asanother example, the distance between the grating 1302D and the closestnanowell of the nanowells associated with the linear waveguide 1306D ishere greater than the distance between the grating 1302C and the closestnanowell of the nanowells associated with the linear waveguide 1306C. Insome implementations, the gratings 1302A and 1302C have the same or asimilar spatial offset. In some implementations, the gratings 1302B and1302D have the same or a similar spatial offset. The characteristic ofthe gratings 1302A-1302D being spatially offset from each other at leastin part facilitates coupling of electromagnetic radiation (e.g., light)into one of the linear waveguides (e.g., the linear waveguide 1306Aand/or 1306C) without coupling the electromagnetic radiation (e.g.,light) into another of the linear waveguides (e.g., the linear waveguide1306B and/or 1306D).

Here, a distance 1308 is less than the resolution distance of theemission optics, and a distance 1310 is greater than, or about equal to,the resolution distance of the emission optics. The distance 1308 hererepresents the separation between the nearest nanowells associated withadjacent linear waveguides. The distance 1310 here represents a distancebetween nanowells associated with the same linear waveguide.

In some implementations, coupling into the gratings 1302A-1302D can alsoor instead be differentiated by a beam parameter other than location ofthe light beam (e.g., but not limited to, angle of incidence,divergence, mode profile, polarization, aspect ratio, diameter,wavelength, and combinations thereof). In some implementations, couplinginto the gratings 1302A-1302D can also or instead be differentiated by acoupler parameter (e.g., but not limited to, grating period, refractiveindex, pitch, groove width, groove height, groove spacing, gratingnon-uniformity, groove orientation, groove curvature, overall shape ofthe coupler, and combinations thereof). In some implementations,coupling into the gratings 1302A-1302D can also or instead bedifferentiated by a waveguide parameter regarding one or more of thelinear waveguides 1306A-1306D (e.g., but not limited to, cross-sectionalprofile, refractive index difference, mode matching with the couplerand/or beam, and combinations thereof).

Examples herein illustrate differential coupling of light into two ormore linear waveguides. Differential coupling can be based on one ormore parameters that characterize the analysis system, the parameter(s)having an effect on the extent to which light is (or is not) coupledinto one or more linear waveguides. In some implementations, one or moresuch parameters can relate to the light beam that is the source ofillumination (e.g., excitation illumination) for the analysis. Forexample, a coupler (e.g., a grating) may be relatively sensitive to oneor more parameters, so a relatively minor change in the parameter(s) canfacilitate differential coupling.

The examples described above illustrate that the flowcell 1300 includesa nanowell layer having first (e.g., the nanowells associated with thelinear waveguide 1306A) and second (e.g., the nanowells associated withthe linear waveguide 1306B) sets of nanowells to receive a sample. Theflowcell 1300 includes a first linear waveguide (e.g., the linearwaveguide 1306A) aligned with the first set of nanowells, and a secondlinear waveguide (e.g., the linear waveguide 1306B) aligned with thesecond set of nanowells; and a first grating (e.g., the grating 1302A)for the first linear waveguide, and a second grating (e.g., the grating1302B) for the second linear waveguide. The first grating has a firstcharacteristic (e.g., being spatially offset from the grating 1302B) tofacilitate coupling of first light into the first linear waveguidewithout coupling the first light into the second linear waveguide.

FIG. 14 schematically shows a light beam 1400 impinging on a surface1402. Examples and/or concepts described with reference to the lightbeam 1400 can be taken into account and/or employed in connection withone or more methods described herein, and/or be used in combination withone or more systems or apparatuses described herein.

The light beam 1400 is here incident at a location 1404 of the surface1402. In some implementations, the location 1404 is a beam parameterthat can be selected and/or adjusted to facilitate differentialcoupling. For example, the location 1404 where the light beam 1400impinges can affect the extent to which light is (or is not) coupledinto one or more linear waveguides.

One or more angles can characterize the incidence of the light beam1400. Here, the light beam 1400 has an angle of incidence 1406 withrespect to a normal of the surface 1402. In some implementations, theangle of incidence 1406 is a beam parameter that can be selected and/oradjusted to facilitate differential coupling. For example, the angle ofincidence 1406 of the light beam 1400 can affect the extent to whichlight is (or is not) coupled into one or more linear waveguides.

One or more characteristics of the light beam 1400 can be taken intoaccount. Here, the light beam 1400 includes individual light rays1400A-1400B that are not parallel to each other but rather form an angle1408 that is a nonzero angle. A divergence of the light beam 1400 can bedefined based on characteristics such as the angle 1408. In someimplementations, the divergence of the light beam 1400 is a beamparameter that can be selected and/or adjusted to facilitatedifferential coupling. For example, the divergence can affect the extentto which light is (or is not) coupled into one or more linearwaveguides.

The light beam 1400 can include coherent light (e.g., a laser beam)which propagates in form of one or more modes. Here, the light beam 1400has a mode profile 1410 that schematically illustrates (e.g., in termsof intensity and/or spatial distribution) the profile of the at leastone mode of the light beam 1400. In some implementations, the modeprofile 1410 is a beam parameter that can be selected and/or adjusted tofacilitate differential coupling. For example, the mode profile 1410 canaffect the extent to which light is (or is not) coupled into one or morelinear waveguides.

The light beam 1400 can have one or more polarizations. In someimplementations, polarization is a beam parameter that can be selectedand/or adjusted to facilitate differential coupling. For example,polarization can affect the extent to which light is (or is not) coupledinto one or more linear waveguides.

The light beam 1400 can have any suitable cross-section profile. In someimplementations, the light beam 1400 has a rectangular cross-sectionprofile 1412A. For example, one or more dimensions of the rectangularcross-section profile 1412A (e.g., an aspect ratio thereof) is a beamparameter that can be selected and/or adjusted to facilitatedifferential coupling. In some implementations, the light beam 1400 hasa circular cross-section profile 1412B. For example, one or moredimensions of the circular cross-section profile 1412B (e.g., a diameterthereof) is a beam parameter that can be selected and/or adjusted tofacilitate differential coupling. The dimension(s) of the rectangularcross-section profile 1412A and/or the circular cross-section profile1412B can affect the extent to which light is (or is not) coupled intoone or more linear waveguides.

The light beam 1400 can include electromagnetic radiation of one or morewavelengths. In some implementations, the wavelength(s) of the lightbeam 1400 is a beam parameter that can be selected and/or adjusted tofacilitate differential coupling. Wavelength(s) can affect the extent towhich light is (or is not) coupled into one or more linear waveguides.For example, different wavelengths couple into a grating at differentangles. A change in the wavelength and the angle of the light beam 1400can allow differential coupling.

In some implementations, one or more parameters affecting differentialcoupling can relate to the grating that couples light into the linearwaveguide for the analysis. For example, a coupler (e.g., a grating) maybe relatively sensitive to one or more parameters, so a relatively minorchange in the parameter(s) can facilitate differential coupling.

FIGS. 15A-15B show examples of gratings 1500 and 1502. The grating 1500and/or 1502 can be used with one or more methods described herein,and/or be used in combination with one or more systems or apparatusesdescribed herein.

The gratings 1500 and 1502 can have the same or different refractiveindices from each other. In some implementations, the refractive indexis a coupler parameter that can be selected and/or adjusted tofacilitate differential coupling. For example, the refractive index canaffect the extent to which light is (or is not) coupled into one or morelinear waveguides.

The grating 1500 here includes grooves 1504, and the grating 1502includes grooves 1506 and 1508. At least one groove pitch 1510 can bedefined for each of the gratings 1500 and 1502. The groove pitch 1510can represent the distance from an edge of one of the grooves 1504, 1506or 1508, to the corresponding edge of the adjacent one of the grooves1504, 1506 or 1508. In some implementations, the groove pitch 1510 is acoupler parameter that can be selected and/or adjusted to facilitatedifferential coupling. For example, the groove pitch 1510 can affect theextent to which light is (or is not) coupled into one or more linearwaveguides.

At least one groove width 1512 can be defined for each of the grooves1504, 1506 or 1508. The groove width 1512 can represent the width fromedge to edge of one of the grooves 1504, 1506 or 1508. In someimplementations, the groove width 1512 is a coupler parameter that canbe selected and/or adjusted to facilitate differential coupling. Forexample, the groove width 1512 can affect the extent to which light is(or is not) coupled into one or more linear waveguides.

At least one groove height 1514 can be defined for each of the grooves1504, 1506 or 1508. The groove height 1514 can represent the height fromthe bottom to the opening of one of the grooves 1504, 1506 or 1508. Insome implementations, the groove height 1514 is a coupler parameter thatcan be selected and/or adjusted to facilitate differential coupling. Forexample, the groove height 1514 can affect the extent to which light is(or is not) coupled into one or more linear waveguides.

At least one groove spacing 1516 can be defined for each of the grooves1504, 1506 or 1508. The groove spacing 1516 can represent the distancefrom the edge of one of the grooves 1504, 1506 or 1508 to the nearestedge of the adjacent one of the grooves 1504, 1506 or 1508. In someimplementations, the groove spacing 1516 is a coupler parameter that canbe selected and/or adjusted to facilitate differential coupling. Forexample, the groove spacing 1516 can affect the extent to which light is(or is not) coupled into one or more linear waveguides.

In some implementations, a non-uniform grating can be used. In someimplementations, the grooves 1506 and 1508 of the grating 1502 provide anon-uniform grating. For example, the grooves 1506 and 1508 may havedifferent groove widths 1512. As another example, the grooves 1506 and1508 may instead or additionally have different groove pitches 1510,different groove heights 1514, and/or different groove spacing 1516. Assuch, the grating 1502 is an example of grating non-uniformity.

In some implementations, groove orientation is a coupler parameter thatcan be selected and/or adjusted to facilitate differential coupling. Insome implementations, gratings generally couple in a transverse electricpolarization in which the electric field is parallel to the gratinggrooves. The grating 1500 and/or 1502 can be positioned so as to obtaina particular orientation of the grooves 1504, 1506 and/or 1508. Forexample, a groove structure can be rotated to another orientation toprovide coupling based on a rotated polarization. That is, the grooveorientation can affect the extent to which light is (or is not) coupledinto one or more linear waveguides.

In some implementations, groove curvature is a coupler parameter thatcan be selected and/or adjusted to facilitate differential coupling.FIG. 15C shows a top view of a grating 1518 with grooves 1520 havingdifferent curvatures. For example, the groove curvature can affect theextent to which light is (or is not) coupled into one or more linearwaveguides.

In some implementations, coupler shape is a coupler parameter that canbe selected and/or adjusted to facilitate differential coupling. FIG. 16shows examples of shapes of couplers 1600, 1602, 1604, and 1606. Theseexamples show illustrative shapes of couplers and schematically indicatethe grooves of the respective gratings. The coupler 1600 can include arectangular (e.g., square) grating. For example, the grooves of thegrating can be oriented along the longer edge, or the shorter edge, ofthe rectangle. The coupler 1602 can include an elliptical (e.g.,circular) grating. For example, the grooves of the grating can beoriented along the major axis, or along the minor axis, of the grating.The coupler 1604 can include a truncated triangular grating. Forexample, the grooves of the grating can be oriented perpendicular to thebase, or perpendicular to the height, of the triangle. As anotherexample, different angles of the side edges can be used. The coupler1606 can include a triangular grating. For example, the grooves of thegrating can be oriented perpendicular to the base, or perpendicular tothe height, of the triangle. As another example, different angles of theside edges can be used. In some implementations, the shape of thecoupler(s) can be selected based on (e.g., optimized) the diameter ofthe illumination beam, or an aspect ratio of the illumination beam, orcombinations thereof, to name just a few examples. The coupler shapeand/or the orientation of the grooves can affect the extent to whichlight is (or is not) coupled into one or more linear waveguides.

The shape of the coupler (including, but not limited to, the couplers1600, 1602, 1604, and 1606) can be selected in view of the diameter,aspect ratio, or other characteristic of the light beam. For example,this can allow the resulting structure to be tuned for a particulardifferential coupling.

The coupler parameter(s) can be selected and/or adjusted based on a modeprofile of the illuminating beam. This can be done by way of selecting(e.g., optimizing) the groove structure. In some implementations, anon-uniform grating can be used. For example, a chirped grating (e.g., agrating with a variation in groove pitch), an apodised grating (e.g.,having a refractive index that approaches zero toward an end of thegrating), a curved grating, and combinations thereof, can be used. Insome implementations, computer-based optimization on one or more couplerparameters (e.g., grating structure) can be performed. For example, thiscan facilitate differential coupling based on the mode profile of theincident light beam.

In some implementations, one or more parameters affecting differentialcoupling can relate to the linear waveguide into which light is coupledfor the analysis. For example, the coupling may be relatively sensitiveto one or more parameters relating to the waveguide, so a relativelyminor change in the parameter(s) can facilitate differential coupling.

In some implementations, a cross-sectional profile of the linearwaveguide is a waveguide parameter that can be selected and/or adjustedto facilitate differential coupling. FIG. 17 shows examples ofcross-sectional profiles for linear waveguides. A waveguide 1700 caninclude a rectangular (e.g., square) profile. For example, a nanowelllayer can be positioned adjacent to the longer edge, or the shorteredge, of the rectangle. A waveguide 1702 can include an elliptical(e.g., circular) profile. For example, the nanowell layer can bepositioned parallel to the major axis, or parallel to the minor axis, ofthe waveguide 1702. A waveguide 1704 can include a truncated triangularprofile. For example, the nanowell layer can be positioned adjacent tothe base, the side edge(s), and/or the truncation face, of the triangle.Different angles of the side edges can be used. A waveguide 1706 caninclude a triangular profile. For example, the nanowell layer can bepositioned adjacent one or more of the sides of the triangle. Differentangles of the side edges can be used. The cross-sectional profile canaffect the extent to which light is (or is not) coupled into one or morelinear waveguides.

In some implementations, the refractive index of the linear waveguide isa waveguide parameter that can be selected and/or adjusted to facilitatedifferential coupling. For example, the refractive index differencebetween two or more linear waveguides can affect the extent to whichlight is (or is not) coupled into the waveguides.

In some implementations, the matching of one or more modes between thelinear waveguide and the coupler, or between the linear waveguide andthe light beam, or both, is a waveguide parameter that can be selectedand/or adjusted to facilitate differential coupling. For example, thedimensions and/or proportions of the linear waveguide can be selected soas to facilitate propagation (or to not facilitate propagation) of aparticular mode of incoming light. That is, mode matching with thecoupler and/or the light beam can affect the extent to which light is(or is not) coupled into the waveguides.

Examples herein mention that a beam parameter, a coupler parameter,and/or a waveguide parameter can be selected and/or adjusted tofacilitate differential coupling. In some implementations, combinationsof two or more such parameters can be selected and/or adjusted. Forexample, the selection/adjustment can take into account at least twobeam parameters; or at least one beam parameter and at least one couplerparameter; or at least one beam parameter, at least one couplerparameter, and at least one waveguide parameter. In someimplementations, a cross-sectional profile of a waveguide can be usedtogether with a particular grating (e.g., a grating optimized forcertain coupling or non-coupling). For example, this can allow theresulting structure to be tuned for different mode profiles, beamdiameters, aspect ratios, to name just a few examples.

FIG. 18 shows a cross section of part of another example flowcell 1800with linear waveguides 1802, 1804 and 1806. The flowcell 1800 can beused with one or more methods described herein, and/or be used incombination with one or more systems or apparatuses described herein.For example, the flowcell 1800 can be used with staggered gratings ornon-staggered gratings, or both. As another example, the flowcell 1800can be used with nanowells arranged in a hexagonal array or anon-hexagonal (e.g., otherwise polygonal) array, or both. Only a portionof the flowcell 1800 is shown, for purposes of illustration. Forexample, one or more additional layers and/or more or fewer waveguides1802, 1804 and/or 1806, can be used.

The flowcell 1800 includes a substrate 1808. The substrate 1808 can forma base for the flowcell 1800. In some implementations, one or more otherlayers can be formed at (e.g., in contact with or near) the substrate1808 in the manufacturing of the flowcell 1800. The substrate 1808 canserve as a basis for forming the linear waveguides 1802, 1804 and/or1806. The linear waveguides 1802, 1804 and/or 1806 can initially existseparately from the substrate 1808 and thereafter be applied onto thesubstrate 1808, or the linear waveguides 1802, 1804 and/or 1806 can beformed by application, and/or removal of, one or more materials to orfrom the substrate. The linear waveguides 1802, 1804 and/or 1806 can beformed directly onto the substrate 1808, or onto one or moreintermediate layers at the substrate 1808.

The linear waveguides 1802, 1804 and/or 1806 serve to conductelectromagnetic radiation (including, but not limited to, visible light,such as laser light). In some implementations, the electromagneticradiation performs one or more functions during an imaging process. Forexample, the electromagnetic radiation can serve to excite fluorophoresin a sample material for imaging. The linear waveguides 1802, 1804and/or 1806 can be made of any suitable material that facilitatespropagation of one or more kinds of electromagnetic radiation. In someimplementations, the material(s) of the linear waveguides 1802, 1804and/or 1806 can include a polymer material. In some implementations, thematerial(s) of the linear waveguides 1802, 1804 and/or 1806 can includeTa₂O₅ and/or SiN_(x). For example, the linear waveguides 1802, 1804and/or 1806 can be formed by sputtering, chemical vapor deposition,atomic layer deposition, spin coating, and/or spray coating.

Each of the linear waveguides 1802, 1804 and/or 1806 can have one ormore gratings (omitted here for clarity) to couple electromagneticradiation into and/or out of that linear waveguide 1802, 1804 and/or1806. The grating(s) can be positioned in the same layer as thecorresponding linear waveguide(s). One or more directions of travel forthe electromagnetic radiation in the linear waveguides 1802, 1804 and/or1806 can be employed. For example, the direction of travel can be intoand/or out of the plane of the present illustration. Examples ofgratings are described elsewhere herein.

Each of the linear waveguides 1802, 1804 and/or 1806 can be positionedagainst one or more types of cladding. The cladding can serve toconstrain the electromagnetic radiation to the respective linearwaveguide 1802, 1804 and/or 1806 and prevent, or reduce the extent of,propagation of the radiation into other linear waveguides 1802, 1804and/or 1806 or other substrates. Here, claddings 1810, 1812, 1814, 1816,and 1818 are shown as an example. In some implementations, the claddings1810, 1812, and 1814, together with the linear waveguides 1802 and 1804,can form a first layer in the flowcell 1800. For example, the claddings1810 and 1812 can be positioned against or near the linear waveguide1802 on different (e.g., opposing) sides thereof. For example, thecladdings 1812 and 1814 can be positioned against or near the linearwaveguide 1804 on different (e.g., opposing) sides thereof. In someimplementations, the claddings 1816 and 1818, together with the linearwaveguide 1806, can form a second layer in the flowcell 1800. Forexample, the claddings 1816 and 1818 can be positioned against or nearthe linear waveguide 1806 on different (e.g., opposing) sides thereof.Formation of multiple layers can provide advantages regardingdifferential coupling. In some implementations, two or more differentmaterials can be used for the respective waveguides. For example, thiscan facilitate that different refractive indices are given to therespective waveguides and/or couplers. In some implementations,cross-talk between waveguides can be reduced or minimized.

The claddings 1810, 1812, 1814, 1816, and/or 1818 can be made from oneor more suitable materials that serve to separate the linear waveguides1802, 1804 and/or 1806 from each other. In some implementations, thecladdings 1810, 1812, 1814, 1816, and/or 1818 can be made from amaterial having a lower refractive index than the refractiveindex/indices of the linear waveguides 1802, 1804 and/or 1806. Forexample, the linear waveguides 1802, 1804 and/or 1806 can have arefractive index of about 1.4-1.6, and the claddings 1810, 1812, 1814,1816, and/or 1818 can have a refractive index of about 1.2-1.4. In someimplementations, one or more of the claddings 1810, 1812, 1814, 1816,and/or 1818 includes a polymer material. In some implementations, one ormore of the claddings 1810, 1812, 1814, 1816, and/or 1818 includesmultiple structures, including, but not limited to, structures of onematerial (e.g., polymer) interspersed by regions of vacuum or anothermaterial (e.g., air or a liquid).

The flowcell 1800 includes at least one nanowell layer 1820. In someimplementations, the nanowell layer 1820 is positioned opposite thefirst layer from the second layer. For example, the nanowell layer 1820can be positioned adjacent (e.g., abutting or near) to the linearwaveguides 1802 and 1804 and the claddings 1810, 1812, and 1814. Thenanowell layer 1820 includes one or more nanowells. In someimplementations, the nanowell layer 1820 includes nanowells 1822, 1824,and 1826. The nanowells 1822, 1824, and/or 1826 can be used for holdingone or more sample materials during at least part of the analysisprocess (e.g., for imaging). For example, one or more genetic materials(e.g., in form of clusters) can be placed in the nanowells 1822, 1824,and/or 1826.

The nanowells 1822, 1824, and/or 1826 can be arranged in any pattern, orwithout a particular pattern, at the nanowell layer 1820. One or more ofthe nanowells 1822, 1824, and/or 1826 can be at least substantiallyaligned with one or more of the linear waveguides 1802, 1804 and/or1806. This can allow interaction between the respective nanowell 1822,1824, and/or 1826 and the corresponding linear waveguide 1802, 1804and/or 1806 for imaging purposes (including, but not limited to, by wayof transmission of evanescent light). For example, the nanowell 1822 canbe at least substantially aligned with the linear waveguide 1802; thenanowell 1824 can be at least substantially aligned with the linearwaveguide 1804; and/or the nanowell 1826 can be at least substantiallyaligned with the linear waveguide 1806. In some implementations, thefirst layer (e.g., the claddings 1810, 1812, and 1814, together with thelinear waveguides 1802 and 1804) can be positioned closer to thenanowell layer 1820 than is the second layer (e.g., the claddings 1816and 1818, together with the linear waveguide 1806). As another example,the second layer can be positioned further from the third layer than isthe first layer.

FIG. 19 is a flowchart of an example method 1900. The method 1900 can beperformed using, and/or in combination with, one or more other examplesdescribed herein. More or fewer operations can be performed, and/or twoor more operations can be performed in a different order, unlessotherwise indicated.

At 1910, a sample is applied to at least some nanowells of a flowcell.In some implementations, the sample is applied to a first set ofnanowells and a second set of nanowells.

At 1920, first light can be differentially coupled into at least a firstlinear waveguide associated with the first set of nanowells. In someimplementations, the first light can be differentially coupled using afirst grating.

At 1930, second light can be differentially coupled into at least asecond linear waveguide associated with the second set of nanowells. Insome implementations, the second light can be differentially coupledusing a second grating.

The terms “substantially” and “about” used throughout this Specificationare used to describe and account for small fluctuations, such as due tovariations in processing. For example, they can refer to less than orequal to ±5%, such as less than or equal to ±2%, such as less than orequal to ±1%, such as less than or equal to ±0.5%, such as less than orequal to ±0.2%, such as less than or equal to ±0.1%, such as less thanor equal to ±0.05%. Also, when used herein, an indefinite article suchas “a” or “an” means “at least one.”

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

A number of implementations have been described. Nevertheless, it willbe understood that various modifications may be made without departingfrom the spirit and scope of the specification.

In addition, the logic flows depicted in the figures do not require theparticular order shown, or sequential order, to achieve desirableresults. In addition, other processes may be provided, or processes maybe eliminated, from the described flows, and other components may beadded to, or removed from, the described systems. Accordingly, otherimplementations are within the scope of the following claims.

While certain features of the described implementations have beenillustrated as described herein, many modifications, substitutions,changes and equivalents will now occur to those skilled in the art. Itis, therefore, to be understood that appended claims are intended tocover all such modifications and changes as fall within the scope of theimplementations. It should be understood that they have been presentedby way of example only, not limitation, and various changes in form anddetails may be made. Any portion of the apparatus and/or methodsdescribed herein may be combined in any combination, except mutuallyexclusive combinations. The implementations described herein can includevarious combinations and/or sub-combinations of the functions,components and/or features of the different implementations described.

What is claimed is:
 1. A flowcell comprising: a substrate; a nanowelllayer having a first set of nanowells and a second set of nanowells toreceive a sample; a first linear waveguide associated with the first setof nanowells; a second linear waveguide associated with the second setof nanowells; a first grating for the first linear waveguide; and asecond grating for the second linear waveguide, the first linearwaveguide and the second linear waveguide positioned between a portionof the substrate and the nanowell layer, wherein one or more of thefirst linear waveguide and the second linear waveguide have a waveguideparameter and wherein one or more of the first grating and the secondgrating have a coupler parameter, the waveguide parameter, the couplerparameter, or both the waveguide parameter and the couple parameterproviding differential coupling of light into the first linear waveguideand the second linear waveguide.
 2. The flowcell of claim 1, wherein thewaveguide parameter comprises at least one of: a cross sectionalprofile, a refractive index difference, a mode matching, or combinationsthereof.
 3. The flowcell of claim 1, wherein the coupler parametercomprises at least one of: a refractive index, a pitch, a groove width,a groove height, a groove spacing, a grating non-uniformity, a grooveorientation, a groove curvature, a coupler shape, or combinationsthereof.
 4. The flowcell of claim 1, wherein at least one of the firstgrating or the second grating is formed by at least one of slits,grooves, ridges, bands, or protruding longitudinal structures.
 5. Theflowcell of claim 1, further comprising first cladding on either side ofthe first grating and second cladding on either side of the secondgrating.
 6. The flowcell of claim 1, wherein the first linear waveguideand the second linear waveguide are formed directly on the substrate. 7.The flowcell of claim 1, further comprising an intermediate layer andwherein the first linear waveguide and the second linear waveguide areformed on the intermediate layer, wherein the intermediate layer ispositioned between the substrate and the first linear waveguide and thesecond linear waveguide.
 8. The flowcell of claim 1, wherein the firstgrating and the second grating are staggered.
 9. The flowcell of claim1, wherein the first set of nanowells comprises a first row of nanowellsthat is aligned with the first linear waveguide and the second set ofnanowells comprises a second row of nanowells that is aligned with thesecond linear waveguide.
 10. The flowcell of claim 1, wherein the firstset of nanowells comprises a first polygonal array of nanowells and thesecond set of nanowells comprises a second polygonal array of nanowells.11. The flowcell of claim 1, wherein the first grating is spatiallyoffset from the second grating in a direction that is substantiallyparallel to the first linear waveguide and the second linear waveguide.12. The flowcell of claim 1, wherein the first grating is to couplefirst light to the first linear waveguide without coupling the firstlight to the second linear waveguide.
 13. The flowcell of claim 1,further comprising a light area aligned with the first grating but notaligned with the second grating.
 14. The flowcell of claim 1, whereinthe first grating and the second grating have different grating periodsfrom each other.
 15. The flowcell of claim 1, wherein the first gratinghas a first refractive index and the second grating has a secondrefractive index.
 16. A flowcell comprising: a nanowell layer having afirst set of nanowells and a second set of nanowells to receive asample; a first linear waveguide array comprising: a plurality of firstlinear waveguides associated with the first set of nanowells; and afirst linear waveguide connector coupled to the first linear waveguides;a second linear waveguide array comprising: a plurality of second linearwaveguides associated with the second set of nanowells; and a secondlinear waveguide connector coupled to the second linear waveguides,wherein during a first scanning operation, light is coupled into thefirst linear waveguides and is not coupled into the second linearwaveguides, and wherein during a second scanning operation, light iscoupled into the second linear waveguides and is not coupled into thefirst linear waveguides.
 17. The flowcell of claim 16, furthercomprising a first linear waveguide component having a first grating anda first coupling component and a second linear waveguide componenthaving a second grating and a second coupling component, the firstlinear waveguide connector connecting the first coupling component andthe first linear waveguides and the second linear waveguide connectorconnecting the second coupling component and the second linearwaveguides.
 18. The flowcell of claim 17, wherein the first linearwaveguide array includes a first linear waveguide distributor coupled tothe first linear waveguide connector and the first linear waveguides andthe second linear waveguide array includes a second linear waveguidedistributor coupled to the second linear waveguide connector and thesecond linear waveguides.
 19. The flowcell of claim 18, wherein thefirst linear waveguides are interspersed between the second linearwaveguides.
 20. The flowcell of claim 17, wherein at least one of thefirst coupling component or the second coupling component comprises asubstrate having a substantially triangular shape.